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Genetics and development
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Genetics and development Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in genetics and development. Current Opinion in Genetics & Development 2000, 10:1–9 Contents (chosen by) 1 Oncogenes and cell proliferation (Mittnacht and Roche) 3 Chromosomes and expression mechanisms (Aasland and
Weinzierl) Genetics of disease (Dawson) Pattern formation and developmental mechanisms (Jones) Differentiation and gene regulation (Schuldt and
5 6 7
Andreazzoli) Genomes and evolution (Douglas)
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• ••
of special interest of outstanding interest
Oncogenes and cell proliferation Selected by Sibylle Mittnacht The Institute of Cancer Research, London, UK
Deregulation of cyclin E induces chromosome instability. Spruck CH, Won K, Reed SI: Nature 1999, 401:297-300. •• Significance: Cyclin E is often over-expressed in tumours. This is not usually associated with increased proliferation activity but correlates with poor prognosis. This paper presents novel insight as to how cyclin E overexpression may promote tumour development. Findings: Using cells engineered to express tetracycline-regulatable cyclin E, the authors show most elegantly that constitutive overexpression of cyclin E leads to chromosome instability. Induction of cyclin E resulted in a significant increase in the proportion of aneuploid cells compared to uninduced controls. This effect was specific to cyclin E and is not seen when other G1/ S cyclins such as cyclins A and D are over-expressed. In tumours, cyclin E may affect the fidelity of chromosome transmission and, through this, exacerbate tumour inherent-gene defects. PPARδδ is an APC-regulated target of nonsteroidal antiinflammatory drugs. He T-C, Chan TA, Vogelstein B, Kinzler KW: Cell 1999, 99:335-345. •• Significance: Evidence has been emerging for some time that painkillers of the aspirin type have preventative effects on colorectal cancer development. This report suggests that aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumourigenesis by inhibiting the DNA-binding activity of the nuclear receptor, PPARδ (peroxisome proliferator antigen receptor δ). This finding could be instrumental for the development of more efficacious and less toxic colorectal cancer chemopreventatives. Findings: The authors identify PPARδ as one of the genes repressed via the adenomatous polyposis coli/β-catenin tumour suppressor pathway. They determine the consensus DNA binding site for this nuclear receptor, which turns out to be distinct
from the known binding site for other members of the PPAR family and demonstrate that NSAIDs directly inhibit binding of PPARδ to this stretch of DNA. Inhibition of telomerase limits the growth of human cancer cells. Hahn WC, Stewardt SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RJ, Knoll JHM, Meyerson M, Weinberg RA. Nat Med 1999, 10:1164-1170. AND
Telomere shortening and apoptosis in telomerase-inhibited human tumour cells. Zhang X, Mar V, Zhou W, Harrington L, Robinson MO: Genes Dev 1999, 13:2388-2399. •• Significance: The telomerase saga continues…. The above reports provide, for the first time, direct evidence that ablation of telomerase activity results in inhibition of cell proliferation and tumour cell death, validating telomerase as a promising drug target for anti-tumour therapeutics. Findings: Hahn et al. demonstrate that constitutive expression of a catalytically impaired telomerase reverse transcriptase (TERT) dominantly represses telomerase activity in tumour cells. Using retroviral transduction, they introduce the mutant TERT into a variety of tumour cells and consistently observe a gradual loss of telomere length, induction of apoptotic death and loss of tumourigenicity. Zhang et al., using an inducible system for expression of a similar TERT mutant, obtain much of the same results. Interestingly, they identify a yet to be explained mechanism by which cells with long telomeres and thus delayed response can reactivate endogenous telomerase activity, undermining the effects of the dominant negative TERT. Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. Gostissa M, Hengsterman A, Fogal V, Sandy P, Schwarz SE, Scheffner M, Del Sal G: EMBO J 1999, 22:6462-6471. AND
SUMO-1 modification activates the transcriptional response of p53. Rodriguez MS, Desterro JMP, Lain S, Midgley CA, Lane D, Hay RT: EMBO J 1999, 22:6455-6461. • Significance: These papers report a novel, regulatory modification of p53 — resulting in stimulation of its ability to activate gene transcription. Findings: Gotissa et al. find the ubiquitin-like protein SUMO-1 to score in a two-hybrid screen with p53 as a bait. They demonstrate that, in cells, p53 can be modified by covalent SUMO-1 attachment and that this modification results in stimulation of p53’s transcriptional activity. In addition to this, Roderiguez et al. demonstrate that SUMO-1 modification of p53 is induced in response to UV irradiation but does not interfere with ubiquitin modification and consequential degradation of p53. This in turn suggests that SUMO-1 modification does not simply operate to stabilize p53 but activate it via an as yet undetermined alternative route. Selected by Serge Roche Centre National de la Recherche Scientifique, Montpellier, France
The p66Shc adaptor controls oxidative stress response and life span in mammals. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, Lanfrancone L, Pelicci PG: Nature 1999, 402:309-313.
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• Significance: An unexpected and specific function for the adapter p66Shc in regulation of life-span and oxidative stress. Findings: The SHC locus encodes three adapter proteins. p46/52Shc mediate the Ras activation induced by mitogens but several reports suggested that the p66Shc was not involved in this pathway. In order to address p66Shc function in vivo, a targeted mutation of the corresponding gene exon was performed in mice. Surprisingly, mutant mice strains showed 30% prolonged life-span and resistance to oxidative stress. In vitro, p66Shc–/–mouse embryo fibroblasts were resistant to apoptosis induced by H2O2 and UV treatment, which correlated with an absence of p53 and p21 upregulation. This process was regulated by serine phosphorylation of p66Shc as the introduction of a serine phosphorylation defective mutant of the protein was unable to restore the stress response in p66Shc–/– cells. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME: Science 1999, 286:1358-1362. AND
The protein kinase p90Rsk as an essential mediator of cytostatic factor activity. Bhatt RR, Ferrell JE Jr: Science 1999, 286:1358-1362. AND
Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Gross SD, Schwab MS, Lewellyn AL, Maller JL: Science 1999, 286:1358-1362. •• Significance: These three papers identify the serine/threonine kinases of the Rsk family as important effectors of the MAP kinase pathway. Findings: Survival of cerebellar granule neurons involved the MAP kinase substrate Rsk2, that phosphorylates and inactivates the proapoptotic protein BAD. Phosphorylation of another Rsk substrate, the transcription factor CREB (cAMP response element binding protein) may also be involved in this process as activated CREB induces cell survival and its inhibition leads to apoptosis (Bonni et al.). A MAP kinase cascade has also been implicated in the metaphase arrest in meiosis II of unfertilized eggs induced by the cytostatic factor (CSF), an event required for preventing parthogenetic activation. Rsk is also an important effector of this signalling cascade: expression of a constitutive active form of Rsk in unfertilized Xenopus laevis eggs mimics the CSF activity (Schwab et al.) and immunodepletion of Rsk in egg extracts prevented the mitotic arrest induced by the MAP kinase cascade which is restored by replenishing the extracts with a competent active Rsk (Bhatt et al.). Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon JE, Pandolfi P, Pavletich NP: Cell 1999, 99:323-334. •• Significance: Structural insight on the phosphoinositide 3phosphatase and tumor supressor phosphatase and tensin homolog deleted on chromosome ten (PTEN). Findings: The crystal structure of a truncated version of human PTEN is reported and reveals a phosphatase domain with an enlarged active site important for phosphoinositide binding and an unexpected C2 domain important for phospholipid membrane binding. A functional C2 region was required for its tumor-suppressor activity as PTEN alleles with reduced affinity
for phospholipids were unable to reverse anchorage-independent growth of glioblastoma cell lines. In addition, both domains of the protein were packed together through an extensive interface found targeted in cancer cells, suggesting an important function of this sequence for PTEN in vivo catalytic activity. Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition. Poy F, Yaffe MB, Sayos J, Saxena K, Morra M, Sumegi J, Cantley LC, Terhorst C, Eck MJ: Mol Cell 1999, 4:555-561. •• Significance: Structural insight on a new class of SH2 domain containing protein with affinity for phospho-tyrosine (pY)-independent peptidic sequence and proposed a mechanism by which these adapters may inhibit specific signaling pathways. Findings: The authors report the three-dimentional structure of the adapter protein SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP). SAP is mainly composed of an SH2 domain that shows affinity with nonphosphorylated peptides in contrast to other SH2 domains. SAP associates with the lymphocyte coreceptor SLAM and peptides of this receptor bind to the SAP-SH2 phosphotyrosine pocket irrespective of its phosphorylation status but it requires a pY amino terminus extended sequence found mutated in XLP patients. A phosphopeptide library screen confirmed that SAP-SH2 has high affinity for TIpYXX(V/I) (X being any amino acid). Interestingly, a similar interaction was detected for the SAP-like protein EAT-2, suggesting that such interactions are not restricted to SAP. The unique feature for these adapters support the notion that they function by negatively regulating the recruitment of SH2-signaling protein to membrane receptors. Interaction of the Ski oncoprotein with Smad3 regulates β signaling. Sun Y, Liu X, Eaton EN, Lane WS, Lodish HF, TGF-β Weinberg RA: Mol Cell 1999, 4:499-509. AND
β signaling by the Negative feedback regulation of TGF-β SnoN oncoprotein. Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K: Science 1999, 286:771-774. AND
c-Ski acts as a transcriptional co-repressor in transforming β signaling through interaction with Smads. growth factor-β Akiyoshi S, Inoue H, Hanai Ji, Kusanagi K, Nemoto N, Miyazono K, Kawabata M: J Biol Chem 1999, 274:35269-35277. •• Significance: The Ski and Sno proto-oncogene products turn out to be negative regulators of the major growth regulatory signaling pathway initiated by TGF-β. This provides strong clues to the mechanism by which Ski oncogenes induce cell transformation. Findings: Smads proteins are transcription activators and important mediators of the TGB-β pathway. In searching for new Smad interactors, an affinity purification approach was used to identify Smad3- (Sun et al.) and Smad4- (Stroschein et al.) associated proteins and a genetic two hybrid approach was also used with Smad 2 (Akiyoshi et al.). With these approaches, the three groups identified proteins of the Ski family. Ski–Smad complex formation was dependent upon TGF-β stimulation and Ski overexpression repressed Smad-dependent transcriptional activation and TGF-β-inhibitory response. Evidence is also provided for a negative regulatory loop of Smads on Ski activity by inducing its protein degradation.
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Control of cell growth by c-Myc in the absence of cell division. Schuhmacher M, Staege MS, Pajic A, Polack A, Weidle UH, Bornkamm GW, Eick D, Kohlhuber F: Curr Biol 1999, 9:1255-1258. AND
c-Myc enhances protein synthesis and cell size during B lymphocyte development. Iritani BM, Eisenman RN: Proc Natl Acad Sci USA 1999, 96:13180-13185. • Significance: An unexpected function for c-Myc in the regulation of cell mass unrelated to cell division in B lymphocytes. Findings: A B cell line that expresses a conditional c-myc allele was used to show that its proliferation depends upon both c-myc expression and the presence of serum in the medium but an observed increase in cell size and protein synthesis was strictly dependent on the presence of c-Myc (Schumacher et al.). Similar observations were reported in vivo using a murine model that expresses a c-myc transgene under the control of the immunoglobulin heavy chain enhancer. These mouse strains displayed increased cell size at all stages of B-cell development and increased cell growth unrelated to the phase of the cell cycle with a two-fold increase in protein synthesis.
IGF-induced MAP kinase activation in the breast cancer epithelial cell line MCF-7. The SRC family protein tyrosine kinase p62Yes controls polymeric IgA transcytosis in vivo. Luton F, Verges M, Vaerman JP, Sudol M, Mostov KE: Mol Cell 1999, 4:627-632. • Significance: A surprising and specific role for the tyrosine kinase cYes of the Src family in epithelial cell function and immunity. Findings: During immune response to an infection, polymeric pIgA is transported through the epithelium to the site of infection. This process involves binding with the polymeric immunoglobulin receptor (pIgR) and transcytosis across the epithelial cells and requires tyrosine kinase activities. The authors report association between the protein tyrosine kinase cYes with pIgR in rodent liver. The functional importance of cYes in this process was supported by a strong reduction in tyrosine kinase activity associated with the receptor in c-yes but not c-src knock out mice, which correlated with an impairment in pIgA transport.
Chromosomes and expression mechanisms Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Pages G, Guerin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P, Pouyssegur J: Science 1999, 286:1374-1377. • Significance: Identification of a specific function for p44MAP kinase in thymocyte maturation. Findings: The p44MAP kinase gene knockout in mice has been performed in order to analyze its function in vivo. No obvious defects were observed suggesting that the loss of p44MAP kinase is compensated by p42 MAP kinase. Nevertheless, a strong defect in thymocyte development was reported with a reduction in proliferation. Interestingly, a sustained p42MAP kinase activation was detected in p44–/– lymphocytes without any compensatory cell response, in agreement with a unique function for p44MAP kinase in these cells.
Differentiation stage-specific inhibition of the Raf-MEKERK pathway by Akt. Rommel C, Clarke BA, Zimmermann S, Nunez L, Rossman R, Reid K, Moelling K, Yancopoulos GD, Glass DJ: Science 1999, 286:1738-1741.
Selected by Rein Aasland University of Bergen, Bergen, Norway
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Okano M, Bell DW, Haber DA, Li E: Cell 1999, 99:247-257. •• Significance: CpG methylation, the methylation of cytosines in CG basepairs, is involved in gene regulation and genome stability in vertebrates. During early development, extensive changes in DNA methylation involves both demetylation and de novo methylation. Although the DNA methyltansferase encoded by Dnmt1 preferentially methylates hemimethylated CpGs, its role in de novo methylation is controversial. Data presented here suggest that the methyltransferases encoded by Dmnt3a and Dnmt3b are primarily responsible for de novo methylation during mouse development. Findings: Dmnt3a–/–Dmnt3b–/– double mutant embryonal stem cell lines were generated and found to be devoid of de novo methylation activity. Double mutant embryos die shortly after gastrulation. The two genes also have non-overlapping functions as Dnmt3b, but not Dnmt3a, was required for certain satellite repeats.
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Phosphorylation and regulation of Raf by Akt. Zimmermann S, Moelling K: Science 1999, 286:1741-1744. • Significance: Cross-talk between two signaling pathways (PI3K-Akt and the Raf-MEK-ERK pathway) depends upon the differention stage of the cell or the cell context. Findings: Both reports identified the serine/threonine kinase Raf as a substrate of the serine/threonine kinase Akt which, in turn, inhibits both Raf activity and the MAP kinase cascade. Rommel et al. observed this inhibitory effect in differentiated myotubes but not in myoblast precursors, suggesting the existence of a molecular event that regulates this cross-talk. Zimmermann and Meolling found a consensus phosphorylation site for Akt in the Raf sequence and reported that indeed Akt phosphorylates and inhibits Raf in vivo when expressed in human embryonic kidney HEK293 cells. Further evidence was shown by the ability of PI3K inhibitor or the expression of a dominant negative form of Akt to enhance
Epigenetic inheritance of active chromatin after removal of the main transactivator. Cavalli G, Paro R: Science 1999, 286:955-958. •• Significance: In Drosphila, the Polycomb (PcG) and trihorax group (trxG) genes are required for maintenance of stable expression patterns of the homeotic genes during embryogenesis. PcG and trxG can act on a regulatory element, called Fab-7, and switch it into either an active or repressive state. It is not known how these states are inherited but stable changes in chromatin structure have been implicated. The data presented here suggest that hyperacetylation of histone H4 can serve such an heritable epigenetic mark. Findings: When a transgene flanked by Fab-7 was transiently activated during early development, it remained active. This activity required trithorax but not Polycomb. The active locus exhibited H4-hyperacetylation. If activated at later stages, the transgene activity and H4-hyperacetylation were only transient.
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mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Ketting RF, Haverkamp THA, van Luenen HGAM, Plasterk RHA: Cell 1999, 99:133-141. • Significance: In Caenorhabditis elegans (and other species), double-stranded RNAs can target genes for post-transcriptional silencing in a phenomenon termed ‘RNA interference’ (RNAi). The RNAi effect can be stable for many cell generations and it can spread throughout the organism. The mechanism for RNAi is not known. Here, a novel RNaseD homolog, MUT-7, is identified as a candidate mRNA exonuclease in RNAi. Findings: The mut-7 gene was isolated in a genetic screen for mutants that are resistant to transposon silencing in the germline. Following the observations described by Tabara et al. [Cell 1999, 99:123-132], mut-7 was tested and found to be resitant to RNAi. Both papers describe additional mutatants that exhibit both phenotypes, suggesting that RNAi and transposon silencing are mechanistically related. A functional enhancer suppresses silencing of a transgene and prevents its localization close to centromeric heterochromatin. Francastel C, Walters MC, Groudine M, Martin DIK: Cell 1999, 99:259-269. • Significance: Enhancers are regulatory elements that potentiate the transcriptional activity of promoters at distance. In the present work, evidence is provided that a functional enhancer can supress silencing of transgenes by preventing their physical association with centromeric heterochromatin. Remarkably, this activity was found to be independent of the transcriptional status of the transgenes. Findings: Wild-type and mutant versions of an enhancer element (5′HS2) of the human β-globin locus were inserted, by means of targeted integration, into three different chromosomal sites, driving a β-galactosidase reporter gene. At a site where a functional enhancer prevented silencing, active transgenes were relocated away from heterochromatin. When a mutant enhancer was placed in this site, the transgene was found close to heterochromatin, both when transcriptionally silent and active. Selected by Robert OJ Weinzierl Imperial College of Science, Technology and Medicine, London, UK
Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Ross-Macdonald P, Coelho PSR, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung K-H, Sheehan A, Symoniatis D, Umansky L et al.: Nature 1999, 402:413-418. •• Significance: The various genome projects are producing a flood of new data that reveal the existence of many new genes with yet unknown functions. This paper describes the first systematic attempt to provide insights into the gene functions, expression patterns and intracellular protein localization on a large scale in the yeast Saccharomyces cerevisiae. Findings: More than 11,000 strains carrying mutations within ∼2,000 different genes were produced by mutagenesis with a specially-engineered mini-transposon. The resulting disruption phenotypes of 7,680 strains were studied under 20 different standardized growth conditions to reveal specific and pleiotropic defects. The phenotypes of transposon insertion in 407 genes was substantially different from wild-type cells and yielded a defined phenotypic characterization of several previously uncharacterized open reading frames. By applying a cre/lox-mediated recombination event — leading to the excision of the majority of
the minitransposon sequences from the insertion site — a number of viable mutants encoding HA-epitope sequence were created. Staining of cells harboring such constructs with fluorescently labelled anti-HA antibodies allowed the visualization of the intracellular location of the encoded gene products.
Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast. Wyrick JJ, Holstege FCP, Jennings EG, Causton HC, Shore D, Grunstein M, Lander ES, Young RA: Nature 1999, 402:418-425. • Significance: This is a high-resolution genome-wide study of the effect of depleting nucleosomes under in vivo conditions. The mRNA levels of 25% of all genes are either up- or downregulated under these conditions but, perhaps surprisingly, the expression of the majority of genes remains mostly unaffected by a 50% reduction of the overall nucleosome packaging density of the genome. Findings: A yeast strain containing only a single copy of the histone H4 gene under the control of a galactose-inducible promoter was grown both in the presence and absence of galactose. mRNAs extracted from these strains were reverse transcribed into fluorescently labelled cDNA to probe a ‘gene chip’ containing all the coding regions of the yeast genome. By comparing the quantities of mRNAs derived from each transcription unit between the strains grown in the presence and absence of galactose it was possible to draw conclusions about the role of nucleosomes in controlling the expression of every coding region present in the genome. A reduction of nucleosome levels to 50% of wild type levels leads to increased expression of 15% of genes and reduced expression of another 10% (more than three-fold in each case). The data suggests a more pervasive role for positive regulation by nucleosomes in yeast than was previously expected.
TFIID-specific yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo. Kormanistky PB, Michel B, Buratowski S: Genes Dev 1999, 13:2484-2489. • Significance: The in vivo functions of TATA binding proteinassociated factors (TAFs; present in the eukaryotic TFIID complex) remain poorly understood. Previous attempts to address this question using yeast genetics have been complicated by the fact that some TAFs are also present as subunits of other transcription complexes. This study describes for the first time the phenotypic consequences of mutations in a TAF exclusively found in TFIID. Findings: Temperature-sensitive alleles of yeast TAF40, a TAF present in TFIID but not in SAGA, were generated by errorprone PCR and introduced into yeast cells by ‘plasmid-shuffling’. Four different mutants exhibiting irreversible growth arrest at elevated temperatures were selected and each found to contain several independently derived point mutations. The phenotypes of the TAF40 mutants could be suppressed by overexpression of TAF19 (their likely interaction partner in TFIID) but not by the overexpression of TATA-binding protein (TBP). At restrictive temperatures, the polyA+-RNA levels dropped sharply in the TAF40 mutants, suggesting that TAFs are required for transcription of most genes in vivo. Significantly, this effect could not be counteracted by the overexpression of TBP within the same cell.
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Genetics of disease Selected by Elisabeth Dawson The Sanger Centre, Cambridge, UK
The DNA sequence of human chromosome 22. Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Brunswick R, Beare DM, Clamp M, Smink LJ et al.: Nature 1999, 402:489-495. •• Significance: The first human chromosome for which the ‘operationally complete’ nucleotide sequence has been established. This provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome and is a unique resource for both genome and medical research. (See also alert by Douglas, p9.) Findings: The sequence of the euchromatic part, the long arm, of human chromosome 22 is reported. The sequenced region consists of 12 contiguous segments spanning 33.4 megabases (Mb), the longest contguous sequence is 23 Mb. Within the sequence there are at least 545 genes and 134 pseudogenes. The sequence is used to compare physical and genetic distances and 4 distinct regions of high recombination are apparent. The average GC content of the chromosome sequence is 47.8%, although the distribution is not uniform. Evidence is shown for the existence of low-copy repeats on chromosome 22; regions of conserved synteny with the mouse genome have been investigated. Association of variant alleles of mannose binding lectin with severity of pulmonary disease in cystic fibrosis: cohort study. Gabolde M, Guilloud-Bataille M, Feingold J, Besmond C: Brit Med J 1999, 319:1166-1167. • Significance: The phenotype of a disease caused by a single gene can be influenced by variations in other modulating genetic factors. Findings: 164 cystic fibrosis patients with homozygous δF508 mutations were assessed for their genotype at the mannose binding lectin gene. Patients who were homozygous or compound heterozygous for mutations of the mannose binding lectin gene had significantly lower measures of lung volume and vital capacity (measures of degradation of pulmonary function). They were also more likely to be harbouring a pulmonary infectious agent, suggesting that individuals at greater risk of pulmonary infection could be more easily identified and specifically cared for. Interindividual variation in mitotic recombination. Holt D, Dreimanis M, Pfeiffer M, Firgaira F, Morley A, Turner D: Am J Hum Genet 1999, 65:1423-1427. • Significance: A study of the variation in the spontaneous frequency of mitotic recombination (MR) in a higher eukaryote. Findings: Mutations can be classified as being caused by intragenic changes, major deletions or MR. MR results in loss of heterozygosity (LOH) in a half of the daughter cells and may have important biological consequences (e.g. LOH is associated with the progression and initiation of some cancers). Using an immunoselection technique, the authors investigated 97 ‘normal’ individuals and showed that the MR mutation frequency in females is significantly greater than that in males (median female:male ratio of 2.5:1). Intragenic mutations frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed it was bimodal with ~10% of individuals showing a very low, or zero, MR. The observed number of individuals from whom no mutants resulting from MR were
obtained was significantly greater than was expected, suggesting that the endogenous level of MR may be under genetic control. Low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply. Human acid α-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II. Bijvoet AGA, van Hirtum H, Kroos MA, van de Kamp EHM, Schoneveld O, Visser P, Brakenhoff JPJ, Weggeman M, van Corven EJ, der Ploeg AT, Reuser AJJ: Hum Mol Genet 1999, 8:2145-2153. • Significance: A successful investigation into a potential enzyme replacement therapy for Pompe’s disease (glycogen storage disease type II). This inherited lysosomal storage disease is fatal, and there is currently no treatment. Findings: Transgenic rabbits were created using a construct containing the human acid α-glucosidase gene and a promotor to effect high expression levels in the milk of the rabbits. RhGAA (recombinant human acid α-glucosidase) was produced at levels of 8g/l milk and was purified for experiments to test its therapeutic effectiveness. Short-term, single-dose experiments in mice deficient for acid α-glucosidase showed full correction of the deficiency in all tissues except the brain. Longer-term experiments, using weekly infusions over a 6 month period resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle together with substantial improvements in tissue morphology. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients. Crooked tail (Cd) models human folate-responsive neural tube defects. Carter M, Ulrich S, Oofuji Y, Williams DA, Ross ME: Hum Mol Genet 1999, 8:2199-2204. • Significance: The genetic animal model used in this study provides a new opportunity to identify the biochemical, genetic and gender-dependent factors contributing to folate-responsive neural-tube defects. Findings: The authors produced intraspecies backcrosses of the Crooked tail (cd) mouse for linkage analysis. cd is a strain that is prone to exencephaly the most common neural tube defect in mice and rats. They mapped cd to a 0.2 cM interval of mouse chromosome 6 and were able to identify cd homozygotes on the basis of genotypes at four closely linked markers. The homozygotes were used to assess the prenatal effects of folic acid supplementation: the number of embryos affected by resorption, early lethality, and exencephaly decreased from 59%→30% when the folic acid supplementation was increased from 0→30 mg/kilogram food. It was also observed that the penetrance of cd depends on the genetic background of the mouse, implying the existence of genetic-modulating factors. Leukaemia disease genes: large-scale cloning and pathway predictions. Li J, Shen H, Himmel KL, Dupuy AJ, Largaespada DA, Nakamura T, Shaughnessy Jr JD, Jenkins NA, Copeland NG: Nat Genet 1999, 23:348-353. •• Significance: A powerful method for the identification of candidate disease genes at increased throughput, using the mouse. The authors demonstrate the effectiveness of the method in leukaemia and identify a number of genes which implicate new disease pathways. Findings: It has previously been noted that retroviral insertion mutagenesis of specific recombinant inbred mice induces a high incidence of leukaemia through activation of oncogenes or disruption of the function of tumour suppressor genes. The
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authors used these mice, and a method based on inverse PCR of proviral genomic sequences, to clone and characterize 419 integration sites. They identified all of the loci previously classified as common integration sites but also revealed a number of new common integration sites which affected loci encoding genes previously implicated in other cancers (e.g. Hras1). In addition, a number of single integration sites were identified in either loci encoding for genes with plausible roles in cancer pathways (e.g. transcription factors, kinases, phosphatases; or in genes which suggested new pathways in the pathogenesis of leukaemia, e.g. cholesterol/lipid metabolism).
mutants. Sato A, Kojima T, Ui-Tei K, Miyata Y, Saigo K: Development 1999, 126:4421-4430. • Significance: Identifies a third member of the frizzled family of wnt receptors in Drospohila and suggests that the gene product may function to attenuate wingless (wg) signaling. Findings: Dfrizzled-3 (Dfz3) expression is similar to and dependent upon wg expression in imaginal discs. Dfz3 binds wg but is not efficient at transducing the wg signal. Hypomorphic alleles of wg revert to near wild-type in the absence of Dfz3, leading to the interpretation that Dfz3 may function as a negative regulator of wg signaling.
Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium: Nature 1999, 401:921-923.
Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node. Levin M, Mercola M: Development 1999, 126:4703-4714. • Significance: The authors suggest that cellular communication between the left and right halves of early chick embryos, via gap junctions, is essential for communicating left–right pattern to Hensen’s node. Findings: Removing either left or right lateral tissues from early stage 2 chick embryos results in abnormal expression of shh and chick nodal, markers normally expressed on the left side. Inhibiting gap junction communication either pharmacologically with lindane, with antisense oligonucleotides, or with antibodies to connexin43 also disrupts normal left–right gene expression. Overall, the results demonstrate a need for cellular communication throughout the early embryo to establish a proper left–right axis.
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Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region. Shiina T, Tamiya G, Oka A, Takishima N, Yamagata T, Kikkawa E, Iwata K, Tomizawa M, Okuaki N, Kuwano Y et al.: Proc Natl Acad Sci USA 1999, 96:13282-13287. •• Significance: The nucleotide sequence of the human MHC region will facilitate positional cloning approaches aimed at defining the molecular basis of HLA-associated diseases. It also provides a new tool for studying the genetics, biology and evolutionary driving forces of human multigene families. Findings: 3.6 Mb of nucleotide sequence representing the human MHC region has been assembled from the work of 4 main groups (The MHC Consortium). Analysis of the sequence revealed the presence of at least 224 genes, of which ∼40% are estimated to have immune function. The paper by Shiina et al. details the sequencing study of a contribution to this whole MHC region, the HLA class I region. Detailed analysis of this region (1.8 Mb of sequence) showed evidence for evolution through repeated segmental duplication and subsequent diversification of a minimal building block. It is also suggested that the non-essential genes, such as HLA-F and MICE have acted as progenitors to today’s immune-competent HLA-A, B, C and MICA/B (evolution by ‘birth and death’).
Pattern formation and developmental mechanisms Selected by Mike Jones Chester Beatty Laboratories, London, UK
β and Lim1 interact in the visceral endoderm to reguHNF3β late primitive streak formation and anterior-posterior polarity in the mouse embryo. Perea-Gomez A, Shawlot W, Sasaki H, Behringer RR, Ang SL: Development 1999, 126:4499-4511. • Significance: Provides evidence that anterior–posterior and dorsal–ventral patterning of the early mouse embryo requires the synergistic functions of HNF3β and Lim1. Findings: HNF3β/Lim1 double mutant embryos exhibit enlarged primitive streaks, which leads to ectopic mesoderm formation. Dorsal mesoderm derivatives are absent, suggesting a defect in mesodermal patterning. Cer-1 and Hex1, markers of the anterior visceral endoderm are absent in double mutants as is the anterior primitive streak marker gsc. Chimeric embryo analysis demonstrates that the function of the two gene products is required in the visceral endoderm. Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic
Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system. Oppenheimer DI, MacNicol AM, Patel NH: Curr Biol 1999, 9:1288-1296. • Significance: A description of the use of a baculovirus-based misexpression system that can be used in species as diverse as insects and amphibia and its application in studying evolutionarily conserved signaling systems. Findings: Recombinant baculoviruses were used to misexpress genes in Drosophila, Tribolium, and Xenopus. Efficient infection and ectopic gene expression was observed in all species. The system was used to demonstrate that the interaction between wingless and engrailed was evolutionarily conserved between organisms as diverse as Drosophila and Tribolium. Polarity of the mouse embryo is anticipated before implantation. Weber RJ, Pedersen RA, Wianny F, Evans MJ, Zernicka-Goetz M: Development 1999, 126:5591-5598. •• Significance: This report provides the first evidence that the polarity of the postimplantation mouse embryo is related to events that occur at least as early as blastocyst stages, before implantation. Findings: Single blastocyst cells were injected with RNA encoding green fluorescent protein (GFP) to serve as a lineage tracer. Cells populating the epiblast became randomly distributed, but those colonising the visceral endoderm (VE) were distributed non-randomly. GFP-positive VE cells originally located near the polar body — a marker of the blastocyst’s axis of symmetry — came to occupy a distal portion of the egg cylinder whereas cells initially located away from the polar body generally took up a proximal (extraembryonic) position. The transformation of the blastocyst axis into the anterior–posterior axis of the embryo is suggested to begin before 5.5 days of gestation.
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Differentiation and gene regulation Selected by Alison Schuldt Wellcome/CRC Institute, Cambridge, UK
Novel functions of nanos in downregulating mitosis and transcription during the development of the Drosophila germline. Deshpande G, Calhoun G, Yanowitz JL, Schedl PD: Cell 1999, 99:271-281. •• Significance: Nanos acts through Sxl to regulate the cell cycle in germ cells and mediate their migration. Nanos additionally acts to repress transcription of several zygotic genes. Findings: Germ cells are distinct from somatic cells in that they are transcriptionally quiescent and have an attenuated cellcycle. Embryos lacking nanos transcribe certain zygotic genes in germ cells. The cell-cycle length of these cells is reduced. Extra germ cells form, which have migration defects, consistent with the proposed role for nanos in germ cell migration. Sxl is necessary and sufficient for these cell cycle and migration defects. Other targets of nanos are proposed to regulate the transcriptional repression of zygotic genes. Differential localisation of two histidine kinases controlling bacterial cell differentiation. Wheeler RT, Shapiro L: Mol Cell 1999, 4:683-694. •• Significance: The asymmetric localisation of the histidine kinases PleC and DivJ is co-ordinated with the cell cycle, demonstrating a novel mechanism for regulating polar differentiation of Caulobacter crescentus. Findings: Green fluorescent protein fusions to PleC and DivJ are followed in living cells and show that these histidine kinases are asymmetrically localised during distinct stages of the C. crescentus cell cycle. They find that PleC is required to mediate the asymmetric localisation of DivJ. This, in turn controls the phosphorylation of DivK — an essential co-ordinating factor in polar differentiation. p27Xic1, a CDK inhibitor, promotes the determination of glial cells in Xenopus retina. Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA: Cell 1999, 99:499-510. •• Significance: The CDK inhibitor p27Xic1 acts with the Notch pathway to induce the Müller glial cell fate, showing that cell cycle plays an active role in cellular determination in the retina. Findings: Overexpression of the CDK inhibitor, p27Xic1 blocks proliferation of all cell types in the devolping retina. It also leads to early differentiation of Müller glial cell type at the expense of neural cell types. Conversely, reduction of p27Xic1 function leads to a loss of Müller cells. The cyclin-binding region of p27Xic1 is responsible for this induction. Cotransfection experiments show that p27Xic1 acts as a determinant in the context of the Notch pathway. Interplay of Notch and FGF signalling restricts cell fate and MAPK activation in the Drosophila trachea. Ikeya T, Hayashi S: Development 1999, 126:4455-4463. • Significance: Notch signalling acts to restrict the fusion cell fate in trachea. It is activated by localised fibroblast growth factor (FGF) signalling between adjacent tissue and trachea through upregulation of Delta. Findings: During tracheal development, a single cell differentiates into a fusion cell at branch tips. Localised FGF signalling between exogenous tissue and trachea stimulates high expression of the Notch receptor, Delta in a single tip cell. Delta, in turn, stimulates Notch signalling in adjacent tip cells while repressing Notch autonomously. Repression of Notch in the
single Delta-expressing cell relieves inhibition of escargot and MAPK, allowing the fusion cell fate to be adopted. Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. Chen CM, Struhl G: Development 1999, 126:5441-5452. • Significance: Frizzled (Fz) and Fz2 function redundantly as the primary receptors for Wingless transduction in Drosophila. Findings: Wingless signalling is lost in the wing imaginal discs of embryos lacking both fz and fz2. Loss of both receptors leads to a loss of Armadillo, leading to the proposal that Fz and Fz2 transduce Wingless signalling through upregulation of Armadillo. Each of these is alone sufficient to confer normal Wingless signalling through development, suggesting that they are functionally redundant. nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Subramaniam K, Seydoux G: Development 1999, 126:4861-4871. • Significance: Two homologs of Drosophila nanos are required for correct differentiation of the primordial germ cells in Caenorhabditis elegans, showing that the molecular mechanisms underlying early germ-cell development is evolutionarily conserved. Findings: In this study, three nanos-related genes — nos-1, nos-2, and nos-3 — are identified. nos-1 and nos-2 are expressed in germ cells and function redundantly during later germline development. nos-1 and nos-2 are required to both maintain germ cell viability post-embryonically and control germ-cell proliferation. RNA-interference experiments suggest that nos-1 and nos-2 may function with a RNA-binding protein related to Pumilio in Drosophila. Selected by Massimiliano Andreazzoli National Institute of Child Health and Human Development, Bethesda, Maryland, USA
Oxygen regulation of airway branching in Drosophila is mediated by Branchless FGF. Jarecki J, Johnson E, Krasnow MA: Cell 1999, 99:211-220. •• Significance: The Drosophila respiratory system consists of primary, secondary and terminal branches. The pattern of terminal branches is remarkably variable, unlike the pattern of primary and secondary branches which is rigidly established. This paper shows that Branchless, (Bnl; Drosophila fibroblast growth factor) acting as a local signal generated by oxygenstarved cells is necessary and sufficient for terminal branching. Thus, Bnl, being also involved in primary and secondary branches specification, appears to be used repeatedly at different levels of trachael branching. Findings: Oxygen deprivation was found to stimulate both Bnl expression and terminal branch sprouting. Ectopic expression experiments demonstrated that Bnl induces terminal branch proliferation whereas expression of a dominant negative form of Bnl receptor was shown to block terminal branching. Mice lacking both presenilin genes exhibit early embryonic patterning defects. Donoviel DB, Hadjantonakis AK, Ikeda M, Zheng H, Hyslop PS, Bernstein A: Genes Dev 1999, 13:28012810. • Significance: Presenilins are cytoplasmic proteins which have been implicated in the positive regulation of the Notch signalling pathway. Combinatorial inactivation of the two murine
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presenilins, PS1 and PS2, demonstrates that these proteins play different but partially overlapping roles in many developmental processes. Findings: Whereas PS1 null mice die perinatally because of several developmental defects, PS2 null mice or PS2 null/PS1 heterozygous mice are normal. Inactivation of one or both copies of PS2 in PS1 null mice, however, gives rise to increasingly severe and pleiotropic defects which might be the consequence of an abnormal mesoderm patterning. Although some aspects of the double null phenotype are similar to the phenotype of mice lacking RBP-Jk, a transcription factor activated by Notch, the widespread defects observed in presenillin null embryos suggest that these proteins might also function outside the Notch pathway. Targeted mutagenesis of the endogenous mouse Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Arango NA, Lovell-Badge R, Behringer RR: Cell 1999, 99:409-419. • Significance: Müllerian inhibiting substance (MIS), a protein secreted by Sertoli cells derived from fetal testis, is essential to cause regression of Mullerian ducts in mammalian males. This work demonstrates the importance of steroidogenic factor 1 (SF1) and SOX9 (a high mobility group protein) binding sites, present in Mis promoter, for gene activity in vivo. Findings: Mutations in SF1 and SOX9 binding sites were introduced by homologous recombination. Whereas mutations in the SF1 site cause a reduction of Mis expression which does not substantially affect sexual development, mutations in the SOX9 site abolish Mis expression resulting in complete retention of Müllerian ducts in male mice. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K, Benezra R: Nature 1999, 401:670-677. •• Significance: Proteins which inhibit DNA binding and cell differentiation (Id proteins) are known to control cell differentiation by heterodimerizing with basic helix-loop-helix (bHLH) transcription factors and thus interfering with their DNA-binding activity. Here, it is shown that Id proteins are required for the correct timing of neuronal differentiation and to support angiogenesis during normal development and in tumour xenografts. Findings: Mice lacking both Id1 and Id3 display a precocious and ectopic expression of neural bHLH genes and an absence of the normal branching and sprouting of capillaries in the neuroectoderm. Intradermal injection of tumour cell lines in mice lacking Id3 and only one copy of Id1 showed a remarkable resistance of these mice to tumour growth as a result of a lack of vascularization of the xenograft. Mesenchymal to epithelial conversion in rat metanephros is induced by LIF. Barasch J, Yang J, Ware CB, Taga T, Yoshida K, Erdjument-Bromage H, Tempst P, Parravicini E, Malach S, Aranoff T, Oliver JA: Cell 1999, 99: 377-386. •• Significance: Signals produced by the ureteric bud are necessary to convert the metanephric mesenchyme to epithelia. This work shows that these signals are mediated by the leukemia inhibitory factor (LIF) and other cytokines of the IL-6 family. Findings: Purification of the epithelializing activity of media conditioned by ureteric bud cells led to the isolation of LIF. A transient exposure to LIF is sufficient to trigger epithelialization of metanephric mesenchyme and subsequent formation of renal
tubules and glomeruli. LIF exerts its effects acting on a cluster of epithelial precursor expressing Pax2 and Wnt4. Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding. Porcher C, Liao EC, Fujiwara Y, Zon LI, Orkin SH: Development 1999, 126:4603-4615. •• Significance: Basic helix-loop-helix (bHLH) transcription factors are thought to regulate cell differentiation through their DNA binding activity. This work demonstrates that stem cell leukemia (SCL), a bHLH factor involved in hematopoiesis and vascular development, is able to exert most of its functions in vivo in a DNA-binding-independent manner. Findings: To define regions of SCL essential for its function, variant forms of the protein were tested for their ability to rescue murine ES cells lacking SCL and the vascular and hematopoietic defects of the zebrafish mutant cloche. The DNA-binding activity of SCL is found to be dispensable to rescue primitive erytropoiesis and to initiate hematopoiesis and vascular development. SCL DNA binding activity is required for the generation of the correct number of erythroid precursor and for erythroid and megakaryocytic precursor maturation.
Genomes and evolution Selected by Susan Douglas Institute for Marine Bioscience, Halifax, Nova Scotia, Canada
A pair of back-to-back papers in Nature describing the sequences of major histocompatibility loci from chicken and human show the dramatic differences in evolutionary strategies employed in the common fight against disease by two groups that diverged from one another 300 million years ago. Complete sequence and gene map of a human major histocompatibility locus. The MHC sequencing consortium: Nature 1999, 401:921-923. •• Significance: A major collaboration from four sequencing groups has revealed 224 gene loci comprising the three classes of mammalian major histocompatability complex (MHC) genes in a 3.6 Mb region of human chromosome 6. As many of these genes are involved in the immune response, this information opens the door to a better understanding of these highly polymorphic genes, their multigene families and the identification of disease loci. Findings: The region of chromosome 6 presented in this study includes the MHC genes in the ‘classical’ Class I, II and III regions as well as the immediate flanking regions which contain additional genes referred to as ‘extended’ Class I and II genes. Immune-related genes comprise ∼40% of the total and are clustered in the ‘classical’ Class II region. Gene density is 1 per 16 kb and pseudogenes abound, particularly in Class I and II regions. Gene duplications have also occurred frequently in these two regions, resulting in new gene family members with distinct functions. The 2% rate of meiotic recombination between the widely-distributed polymorphic genes has resulted in a huge variety of haplotypes in humans. The chicken B locus is a minimal essential major histocompatibility locus. Kaufman J, Milne S, Göbel TWF, Walker BA, Jacob JP, Auffray C, Zoorob R, Beck S: Nature 1999, 401:923-925. •• Significance: Sequence analysis of the 19 genes present in a 92 kb region of the BF/BL region of the chicken B locus
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shows both striking similarities and remarkable contrasts to its human MHC counterpart. The chicken MHC exhibits a strong association with resistance and susceptibility to certain infectious pathogens, and thus provides a simple model for investigating the molecular basis for MHC-determined pathogen-specific disease resistance. Findings: Although most of the genes in the chicken locus have counterparts in the human MHC gene classes, they are organized differently and much more compactly — short introns and intergenic regions, few repeats, no pseudogenes. Furthermore, genes expected to be present (e.g. proteasome components) are absent and genes not found in human MHC (e.g. natural killer [NK] receptor, are present). The compressed structure of the chicken MHC allows co-evolution of genes resulting in haplotypes that are relatively stable over evolutionary time.
The DNA sequence of human chromosome 22. Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Brunswick R, Beare DM, Clamp M, Smink LJ et al.: Nature 1999, 402:489-495. •• Significance: This landmark paper from the Human Genome Project (HGP) presents the first glimpse of a complete human chromosome and provides insights into the information to be gleaned from the remaining chromosomes. Chromosome 22 is the second smallest autosomal chromosome and is one of five human acrocentric chromosomes. Its gene content is rich and it contains many regions associated with human disease including schizophrenia and spinocerebellar ataxia. The sequence provides a model for the testing of computational tools for gene prediction and repeat element recognition. (See also alert by Dawson, p5.) Findings: The 33.4 Mb of sequence was obtained using the HGP ‘clone by clone’ approach and is presented as 12 contiguous segments separated by gaps of <150 kb that were intractable to sequencing using available cloning systems. The 545 annotated genes and 134 pseudogenes comprise 39% of the total sequence and exhibit a wide range in size from 1 kb to >583 kb. Tandem and interspersed repeat sequences comprise 41.9% of the genome. A number of duplicated gene families and low-copy repeats have been identified. Physical locations of microsatellite markers have been assigned to the chromosome and regions differing dramatically in G+C content (isochores) have been mapped. Comparison with the mouse genome reveals 8 regions of conserved synteny and allows sites of evolutionary rearrangements to be identified accurately at the nucleotide level.
Novel coding regions in four complete archaeal genomes. Raghavan S, Ouzounis CA: Nucleic Acids Res 1999, 27:4405-4408. • Significance: Prediction of coding sequences from genomes is not always perfect as a result of incomplete annotation, sequencing errors and the limitations in functional information of many genes. Understandably, many ‘non-coding’ regions may therefore actually contain potential protein-coding regions that have escaped detection. This paper compares all of the regions between annotated open reading frames (ORFs) in each of four completely sequenced archaeal genomes in an attempt to identify potential coding regions. Findings: 2237 inter-ORF regions from the complete genomes of Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus and Pyrococcus horikoshii were used to query the non-redundant protein sequence database using BlastX v. 2.0. 524 regions contained complete ORFs (113) and sections of previously characterised ORFs with conflicting start/end sites (411). The closest homologs of most of the newly identified ORFs were other archaeal proteins. In 21 cases, the ORF had similarity to a protein of known function. This finding underscores the importance of regularly matching ‘non-coding regions’ against protein databases. The distribution of RNA motifs in natural sequences. Bourdeau V, Ferbeyre G, Pageau M, Paquin B, Cedergren R: Nucleic Acids Res 1999, 27:4457-4467. • Significance: The identification of RNA genes and structures in sequenced genomes has not been given the attention that their protein-coding counterparts have. This paper describes the search for such RNA structures using motifs and introduces the concept of ‘ribonomics’. The motifs are broadly categorized as protein-binding, chemically and catalytically active, and small molecule-binding. The origin of motifs and the significance of their wide distribution in the database can now be discussed and the knowledge used in logical RNA based-intervention for modulating biological processes. Findings: Using the computer search engine RNAMOT, searches of both strands of sequence data in GenBank as well as a random sequence database of 10,000 sequences of 100,000 nucleotides each were carried out. The origin of the majority of motifs by random evolutionary drift is supported by comparison of the distribution of motifs in natural sequences versus random sequences. An RNA motif database comprising the distribution of motifs among 67 GenBank features as well as their phylogenetic distribution was constructed and is available at http://www.centrcn.umontreal.ca/bourdeav/Ribonomics
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Genetics and development Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in genetics and development. Current Opinion in Genetics & Development 2000, 10:1–9 Contents (chosen by) 1 Oncogenes and cell proliferation (Mittnacht and Roche) 3 Chromosomes and expression mechanisms (Aasland and
Weinzierl) Genetics of disease (Dawson) Pattern formation and developmental mechanisms (Jones) Differentiation and gene regulation (Schuldt and
5 6 7
Andreazzoli) Genomes and evolution (Douglas)
8
• ••
of special interest of outstanding interest
Oncogenes and cell proliferation Selected by Sibylle Mittnacht The Institute of Cancer Research, London, UK
Deregulation of cyclin E induces chromosome instability. Spruck CH, Won K, Reed SI: Nature 1999, 401:297-300. •• Significance: Cyclin E is often over-expressed in tumours. This is not usually associated with increased proliferation activity but correlates with poor prognosis. This paper presents novel insight as to how cyclin E overexpression may promote tumour development. Findings: Using cells engineered to express tetracycline-regulatable cyclin E, the authors show most elegantly that constitutive overexpression of cyclin E leads to chromosome instability. Induction of cyclin E resulted in a significant increase in the proportion of aneuploid cells compared to uninduced controls. This effect was specific to cyclin E and is not seen when other G1/ S cyclins such as cyclins A and D are over-expressed. In tumours, cyclin E may affect the fidelity of chromosome transmission and, through this, exacerbate tumour inherent-gene defects. PPARδδ is an APC-regulated target of nonsteroidal antiinflammatory drugs. He T-C, Chan TA, Vogelstein B, Kinzler KW: Cell 1999, 99:335-345. •• Significance: Evidence has been emerging for some time that painkillers of the aspirin type have preventative effects on colorectal cancer development. This report suggests that aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumourigenesis by inhibiting the DNA-binding activity of the nuclear receptor, PPARδ (peroxisome proliferator antigen receptor δ). This finding could be instrumental for the development of more efficacious and less toxic colorectal cancer chemopreventatives. Findings: The authors identify PPARδ as one of the genes repressed via the adenomatous polyposis coli/β-catenin tumour suppressor pathway. They determine the consensus DNA binding site for this nuclear receptor, which turns out to be distinct
from the known binding site for other members of the PPAR family and demonstrate that NSAIDs directly inhibit binding of PPARδ to this stretch of DNA. Inhibition of telomerase limits the growth of human cancer cells. Hahn WC, Stewardt SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RJ, Knoll JHM, Meyerson M, Weinberg RA. Nat Med 1999, 10:1164-1170. AND
Telomere shortening and apoptosis in telomerase-inhibited human tumour cells. Zhang X, Mar V, Zhou W, Harrington L, Robinson MO: Genes Dev 1999, 13:2388-2399. •• Significance: The telomerase saga continues…. The above reports provide, for the first time, direct evidence that ablation of telomerase activity results in inhibition of cell proliferation and tumour cell death, validating telomerase as a promising drug target for anti-tumour therapeutics. Findings: Hahn et al. demonstrate that constitutive expression of a catalytically impaired telomerase reverse transcriptase (TERT) dominantly represses telomerase activity in tumour cells. Using retroviral transduction, they introduce the mutant TERT into a variety of tumour cells and consistently observe a gradual loss of telomere length, induction of apoptotic death and loss of tumourigenicity. Zhang et al., using an inducible system for expression of a similar TERT mutant, obtain much of the same results. Interestingly, they identify a yet to be explained mechanism by which cells with long telomeres and thus delayed response can reactivate endogenous telomerase activity, undermining the effects of the dominant negative TERT. Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. Gostissa M, Hengsterman A, Fogal V, Sandy P, Schwarz SE, Scheffner M, Del Sal G: EMBO J 1999, 22:6462-6471. AND
SUMO-1 modification activates the transcriptional response of p53. Rodriguez MS, Desterro JMP, Lain S, Midgley CA, Lane D, Hay RT: EMBO J 1999, 22:6455-6461. • Significance: These papers report a novel, regulatory modification of p53 — resulting in stimulation of its ability to activate gene transcription. Findings: Gotissa et al. find the ubiquitin-like protein SUMO-1 to score in a two-hybrid screen with p53 as a bait. They demonstrate that, in cells, p53 can be modified by covalent SUMO-1 attachment and that this modification results in stimulation of p53’s transcriptional activity. In addition to this, Roderiguez et al. demonstrate that SUMO-1 modification of p53 is induced in response to UV irradiation but does not interfere with ubiquitin modification and consequential degradation of p53. This in turn suggests that SUMO-1 modification does not simply operate to stabilize p53 but activate it via an as yet undetermined alternative route. Selected by Serge Roche Centre National de la Recherche Scientifique, Montpellier, France
The p66Shc adaptor controls oxidative stress response and life span in mammals. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, Lanfrancone L, Pelicci PG: Nature 1999, 402:309-313.
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• Significance: An unexpected and specific function for the adapter p66Shc in regulation of life-span and oxidative stress. Findings: The SHC locus encodes three adapter proteins. p46/52Shc mediate the Ras activation induced by mitogens but several reports suggested that the p66Shc was not involved in this pathway. In order to address p66Shc function in vivo, a targeted mutation of the corresponding gene exon was performed in mice. Surprisingly, mutant mice strains showed 30% prolonged life-span and resistance to oxidative stress. In vitro, p66Shc–/–mouse embryo fibroblasts were resistant to apoptosis induced by H2O2 and UV treatment, which correlated with an absence of p53 and p21 upregulation. This process was regulated by serine phosphorylation of p66Shc as the introduction of a serine phosphorylation defective mutant of the protein was unable to restore the stress response in p66Shc–/– cells. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME: Science 1999, 286:1358-1362. AND
The protein kinase p90Rsk as an essential mediator of cytostatic factor activity. Bhatt RR, Ferrell JE Jr: Science 1999, 286:1358-1362. AND
Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Gross SD, Schwab MS, Lewellyn AL, Maller JL: Science 1999, 286:1358-1362. •• Significance: These three papers identify the serine/threonine kinases of the Rsk family as important effectors of the MAP kinase pathway. Findings: Survival of cerebellar granule neurons involved the MAP kinase substrate Rsk2, that phosphorylates and inactivates the proapoptotic protein BAD. Phosphorylation of another Rsk substrate, the transcription factor CREB (cAMP response element binding protein) may also be involved in this process as activated CREB induces cell survival and its inhibition leads to apoptosis (Bonni et al.). A MAP kinase cascade has also been implicated in the metaphase arrest in meiosis II of unfertilized eggs induced by the cytostatic factor (CSF), an event required for preventing parthogenetic activation. Rsk is also an important effector of this signalling cascade: expression of a constitutive active form of Rsk in unfertilized Xenopus laevis eggs mimics the CSF activity (Schwab et al.) and immunodepletion of Rsk in egg extracts prevented the mitotic arrest induced by the MAP kinase cascade which is restored by replenishing the extracts with a competent active Rsk (Bhatt et al.). Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon JE, Pandolfi P, Pavletich NP: Cell 1999, 99:323-334. •• Significance: Structural insight on the phosphoinositide 3phosphatase and tumor supressor phosphatase and tensin homolog deleted on chromosome ten (PTEN). Findings: The crystal structure of a truncated version of human PTEN is reported and reveals a phosphatase domain with an enlarged active site important for phosphoinositide binding and an unexpected C2 domain important for phospholipid membrane binding. A functional C2 region was required for its tumor-suppressor activity as PTEN alleles with reduced affinity
for phospholipids were unable to reverse anchorage-independent growth of glioblastoma cell lines. In addition, both domains of the protein were packed together through an extensive interface found targeted in cancer cells, suggesting an important function of this sequence for PTEN in vivo catalytic activity. Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition. Poy F, Yaffe MB, Sayos J, Saxena K, Morra M, Sumegi J, Cantley LC, Terhorst C, Eck MJ: Mol Cell 1999, 4:555-561. •• Significance: Structural insight on a new class of SH2 domain containing protein with affinity for phospho-tyrosine (pY)-independent peptidic sequence and proposed a mechanism by which these adapters may inhibit specific signaling pathways. Findings: The authors report the three-dimentional structure of the adapter protein SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP). SAP is mainly composed of an SH2 domain that shows affinity with nonphosphorylated peptides in contrast to other SH2 domains. SAP associates with the lymphocyte coreceptor SLAM and peptides of this receptor bind to the SAP-SH2 phosphotyrosine pocket irrespective of its phosphorylation status but it requires a pY amino terminus extended sequence found mutated in XLP patients. A phosphopeptide library screen confirmed that SAP-SH2 has high affinity for TIpYXX(V/I) (X being any amino acid). Interestingly, a similar interaction was detected for the SAP-like protein EAT-2, suggesting that such interactions are not restricted to SAP. The unique feature for these adapters support the notion that they function by negatively regulating the recruitment of SH2-signaling protein to membrane receptors. Interaction of the Ski oncoprotein with Smad3 regulates β signaling. Sun Y, Liu X, Eaton EN, Lane WS, Lodish HF, TGF-β Weinberg RA: Mol Cell 1999, 4:499-509. AND
β signaling by the Negative feedback regulation of TGF-β SnoN oncoprotein. Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K: Science 1999, 286:771-774. AND
c-Ski acts as a transcriptional co-repressor in transforming β signaling through interaction with Smads. growth factor-β Akiyoshi S, Inoue H, Hanai Ji, Kusanagi K, Nemoto N, Miyazono K, Kawabata M: J Biol Chem 1999, 274:35269-35277. •• Significance: The Ski and Sno proto-oncogene products turn out to be negative regulators of the major growth regulatory signaling pathway initiated by TGF-β. This provides strong clues to the mechanism by which Ski oncogenes induce cell transformation. Findings: Smads proteins are transcription activators and important mediators of the TGB-β pathway. In searching for new Smad interactors, an affinity purification approach was used to identify Smad3- (Sun et al.) and Smad4- (Stroschein et al.) associated proteins and a genetic two hybrid approach was also used with Smad 2 (Akiyoshi et al.). With these approaches, the three groups identified proteins of the Ski family. Ski–Smad complex formation was dependent upon TGF-β stimulation and Ski overexpression repressed Smad-dependent transcriptional activation and TGF-β-inhibitory response. Evidence is also provided for a negative regulatory loop of Smads on Ski activity by inducing its protein degradation.
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Control of cell growth by c-Myc in the absence of cell division. Schuhmacher M, Staege MS, Pajic A, Polack A, Weidle UH, Bornkamm GW, Eick D, Kohlhuber F: Curr Biol 1999, 9:1255-1258. AND
c-Myc enhances protein synthesis and cell size during B lymphocyte development. Iritani BM, Eisenman RN: Proc Natl Acad Sci USA 1999, 96:13180-13185. • Significance: An unexpected function for c-Myc in the regulation of cell mass unrelated to cell division in B lymphocytes. Findings: A B cell line that expresses a conditional c-myc allele was used to show that its proliferation depends upon both c-myc expression and the presence of serum in the medium but an observed increase in cell size and protein synthesis was strictly dependent on the presence of c-Myc (Schumacher et al.). Similar observations were reported in vivo using a murine model that expresses a c-myc transgene under the control of the immunoglobulin heavy chain enhancer. These mouse strains displayed increased cell size at all stages of B-cell development and increased cell growth unrelated to the phase of the cell cycle with a two-fold increase in protein synthesis.
IGF-induced MAP kinase activation in the breast cancer epithelial cell line MCF-7. The SRC family protein tyrosine kinase p62Yes controls polymeric IgA transcytosis in vivo. Luton F, Verges M, Vaerman JP, Sudol M, Mostov KE: Mol Cell 1999, 4:627-632. • Significance: A surprising and specific role for the tyrosine kinase cYes of the Src family in epithelial cell function and immunity. Findings: During immune response to an infection, polymeric pIgA is transported through the epithelium to the site of infection. This process involves binding with the polymeric immunoglobulin receptor (pIgR) and transcytosis across the epithelial cells and requires tyrosine kinase activities. The authors report association between the protein tyrosine kinase cYes with pIgR in rodent liver. The functional importance of cYes in this process was supported by a strong reduction in tyrosine kinase activity associated with the receptor in c-yes but not c-src knock out mice, which correlated with an impairment in pIgA transport.
Chromosomes and expression mechanisms Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Pages G, Guerin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P, Pouyssegur J: Science 1999, 286:1374-1377. • Significance: Identification of a specific function for p44MAP kinase in thymocyte maturation. Findings: The p44MAP kinase gene knockout in mice has been performed in order to analyze its function in vivo. No obvious defects were observed suggesting that the loss of p44MAP kinase is compensated by p42 MAP kinase. Nevertheless, a strong defect in thymocyte development was reported with a reduction in proliferation. Interestingly, a sustained p42MAP kinase activation was detected in p44–/– lymphocytes without any compensatory cell response, in agreement with a unique function for p44MAP kinase in these cells.
Differentiation stage-specific inhibition of the Raf-MEKERK pathway by Akt. Rommel C, Clarke BA, Zimmermann S, Nunez L, Rossman R, Reid K, Moelling K, Yancopoulos GD, Glass DJ: Science 1999, 286:1738-1741.
Selected by Rein Aasland University of Bergen, Bergen, Norway
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Okano M, Bell DW, Haber DA, Li E: Cell 1999, 99:247-257. •• Significance: CpG methylation, the methylation of cytosines in CG basepairs, is involved in gene regulation and genome stability in vertebrates. During early development, extensive changes in DNA methylation involves both demetylation and de novo methylation. Although the DNA methyltansferase encoded by Dnmt1 preferentially methylates hemimethylated CpGs, its role in de novo methylation is controversial. Data presented here suggest that the methyltransferases encoded by Dmnt3a and Dnmt3b are primarily responsible for de novo methylation during mouse development. Findings: Dmnt3a–/–Dmnt3b–/– double mutant embryonal stem cell lines were generated and found to be devoid of de novo methylation activity. Double mutant embryos die shortly after gastrulation. The two genes also have non-overlapping functions as Dnmt3b, but not Dnmt3a, was required for certain satellite repeats.
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Phosphorylation and regulation of Raf by Akt. Zimmermann S, Moelling K: Science 1999, 286:1741-1744. • Significance: Cross-talk between two signaling pathways (PI3K-Akt and the Raf-MEK-ERK pathway) depends upon the differention stage of the cell or the cell context. Findings: Both reports identified the serine/threonine kinase Raf as a substrate of the serine/threonine kinase Akt which, in turn, inhibits both Raf activity and the MAP kinase cascade. Rommel et al. observed this inhibitory effect in differentiated myotubes but not in myoblast precursors, suggesting the existence of a molecular event that regulates this cross-talk. Zimmermann and Meolling found a consensus phosphorylation site for Akt in the Raf sequence and reported that indeed Akt phosphorylates and inhibits Raf in vivo when expressed in human embryonic kidney HEK293 cells. Further evidence was shown by the ability of PI3K inhibitor or the expression of a dominant negative form of Akt to enhance
Epigenetic inheritance of active chromatin after removal of the main transactivator. Cavalli G, Paro R: Science 1999, 286:955-958. •• Significance: In Drosphila, the Polycomb (PcG) and trihorax group (trxG) genes are required for maintenance of stable expression patterns of the homeotic genes during embryogenesis. PcG and trxG can act on a regulatory element, called Fab-7, and switch it into either an active or repressive state. It is not known how these states are inherited but stable changes in chromatin structure have been implicated. The data presented here suggest that hyperacetylation of histone H4 can serve such an heritable epigenetic mark. Findings: When a transgene flanked by Fab-7 was transiently activated during early development, it remained active. This activity required trithorax but not Polycomb. The active locus exhibited H4-hyperacetylation. If activated at later stages, the transgene activity and H4-hyperacetylation were only transient.
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mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Ketting RF, Haverkamp THA, van Luenen HGAM, Plasterk RHA: Cell 1999, 99:133-141. • Significance: In Caenorhabditis elegans (and other species), double-stranded RNAs can target genes for post-transcriptional silencing in a phenomenon termed ‘RNA interference’ (RNAi). The RNAi effect can be stable for many cell generations and it can spread throughout the organism. The mechanism for RNAi is not known. Here, a novel RNaseD homolog, MUT-7, is identified as a candidate mRNA exonuclease in RNAi. Findings: The mut-7 gene was isolated in a genetic screen for mutants that are resistant to transposon silencing in the germline. Following the observations described by Tabara et al. [Cell 1999, 99:123-132], mut-7 was tested and found to be resitant to RNAi. Both papers describe additional mutatants that exhibit both phenotypes, suggesting that RNAi and transposon silencing are mechanistically related. A functional enhancer suppresses silencing of a transgene and prevents its localization close to centromeric heterochromatin. Francastel C, Walters MC, Groudine M, Martin DIK: Cell 1999, 99:259-269. • Significance: Enhancers are regulatory elements that potentiate the transcriptional activity of promoters at distance. In the present work, evidence is provided that a functional enhancer can supress silencing of transgenes by preventing their physical association with centromeric heterochromatin. Remarkably, this activity was found to be independent of the transcriptional status of the transgenes. Findings: Wild-type and mutant versions of an enhancer element (5′HS2) of the human β-globin locus were inserted, by means of targeted integration, into three different chromosomal sites, driving a β-galactosidase reporter gene. At a site where a functional enhancer prevented silencing, active transgenes were relocated away from heterochromatin. When a mutant enhancer was placed in this site, the transgene was found close to heterochromatin, both when transcriptionally silent and active. Selected by Robert OJ Weinzierl Imperial College of Science, Technology and Medicine, London, UK
Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Ross-Macdonald P, Coelho PSR, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung K-H, Sheehan A, Symoniatis D, Umansky L et al.: Nature 1999, 402:413-418. •• Significance: The various genome projects are producing a flood of new data that reveal the existence of many new genes with yet unknown functions. This paper describes the first systematic attempt to provide insights into the gene functions, expression patterns and intracellular protein localization on a large scale in the yeast Saccharomyces cerevisiae. Findings: More than 11,000 strains carrying mutations within ∼2,000 different genes were produced by mutagenesis with a specially-engineered mini-transposon. The resulting disruption phenotypes of 7,680 strains were studied under 20 different standardized growth conditions to reveal specific and pleiotropic defects. The phenotypes of transposon insertion in 407 genes was substantially different from wild-type cells and yielded a defined phenotypic characterization of several previously uncharacterized open reading frames. By applying a cre/lox-mediated recombination event — leading to the excision of the majority of
the minitransposon sequences from the insertion site — a number of viable mutants encoding HA-epitope sequence were created. Staining of cells harboring such constructs with fluorescently labelled anti-HA antibodies allowed the visualization of the intracellular location of the encoded gene products.
Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast. Wyrick JJ, Holstege FCP, Jennings EG, Causton HC, Shore D, Grunstein M, Lander ES, Young RA: Nature 1999, 402:418-425. • Significance: This is a high-resolution genome-wide study of the effect of depleting nucleosomes under in vivo conditions. The mRNA levels of 25% of all genes are either up- or downregulated under these conditions but, perhaps surprisingly, the expression of the majority of genes remains mostly unaffected by a 50% reduction of the overall nucleosome packaging density of the genome. Findings: A yeast strain containing only a single copy of the histone H4 gene under the control of a galactose-inducible promoter was grown both in the presence and absence of galactose. mRNAs extracted from these strains were reverse transcribed into fluorescently labelled cDNA to probe a ‘gene chip’ containing all the coding regions of the yeast genome. By comparing the quantities of mRNAs derived from each transcription unit between the strains grown in the presence and absence of galactose it was possible to draw conclusions about the role of nucleosomes in controlling the expression of every coding region present in the genome. A reduction of nucleosome levels to 50% of wild type levels leads to increased expression of 15% of genes and reduced expression of another 10% (more than three-fold in each case). The data suggests a more pervasive role for positive regulation by nucleosomes in yeast than was previously expected.
TFIID-specific yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo. Kormanistky PB, Michel B, Buratowski S: Genes Dev 1999, 13:2484-2489. • Significance: The in vivo functions of TATA binding proteinassociated factors (TAFs; present in the eukaryotic TFIID complex) remain poorly understood. Previous attempts to address this question using yeast genetics have been complicated by the fact that some TAFs are also present as subunits of other transcription complexes. This study describes for the first time the phenotypic consequences of mutations in a TAF exclusively found in TFIID. Findings: Temperature-sensitive alleles of yeast TAF40, a TAF present in TFIID but not in SAGA, were generated by errorprone PCR and introduced into yeast cells by ‘plasmid-shuffling’. Four different mutants exhibiting irreversible growth arrest at elevated temperatures were selected and each found to contain several independently derived point mutations. The phenotypes of the TAF40 mutants could be suppressed by overexpression of TAF19 (their likely interaction partner in TFIID) but not by the overexpression of TATA-binding protein (TBP). At restrictive temperatures, the polyA+-RNA levels dropped sharply in the TAF40 mutants, suggesting that TAFs are required for transcription of most genes in vivo. Significantly, this effect could not be counteracted by the overexpression of TBP within the same cell.
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Genetics of disease Selected by Elisabeth Dawson The Sanger Centre, Cambridge, UK
The DNA sequence of human chromosome 22. Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Brunswick R, Beare DM, Clamp M, Smink LJ et al.: Nature 1999, 402:489-495. •• Significance: The first human chromosome for which the ‘operationally complete’ nucleotide sequence has been established. This provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome and is a unique resource for both genome and medical research. (See also alert by Douglas, p9.) Findings: The sequence of the euchromatic part, the long arm, of human chromosome 22 is reported. The sequenced region consists of 12 contiguous segments spanning 33.4 megabases (Mb), the longest contguous sequence is 23 Mb. Within the sequence there are at least 545 genes and 134 pseudogenes. The sequence is used to compare physical and genetic distances and 4 distinct regions of high recombination are apparent. The average GC content of the chromosome sequence is 47.8%, although the distribution is not uniform. Evidence is shown for the existence of low-copy repeats on chromosome 22; regions of conserved synteny with the mouse genome have been investigated. Association of variant alleles of mannose binding lectin with severity of pulmonary disease in cystic fibrosis: cohort study. Gabolde M, Guilloud-Bataille M, Feingold J, Besmond C: Brit Med J 1999, 319:1166-1167. • Significance: The phenotype of a disease caused by a single gene can be influenced by variations in other modulating genetic factors. Findings: 164 cystic fibrosis patients with homozygous δF508 mutations were assessed for their genotype at the mannose binding lectin gene. Patients who were homozygous or compound heterozygous for mutations of the mannose binding lectin gene had significantly lower measures of lung volume and vital capacity (measures of degradation of pulmonary function). They were also more likely to be harbouring a pulmonary infectious agent, suggesting that individuals at greater risk of pulmonary infection could be more easily identified and specifically cared for. Interindividual variation in mitotic recombination. Holt D, Dreimanis M, Pfeiffer M, Firgaira F, Morley A, Turner D: Am J Hum Genet 1999, 65:1423-1427. • Significance: A study of the variation in the spontaneous frequency of mitotic recombination (MR) in a higher eukaryote. Findings: Mutations can be classified as being caused by intragenic changes, major deletions or MR. MR results in loss of heterozygosity (LOH) in a half of the daughter cells and may have important biological consequences (e.g. LOH is associated with the progression and initiation of some cancers). Using an immunoselection technique, the authors investigated 97 ‘normal’ individuals and showed that the MR mutation frequency in females is significantly greater than that in males (median female:male ratio of 2.5:1). Intragenic mutations frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed it was bimodal with ~10% of individuals showing a very low, or zero, MR. The observed number of individuals from whom no mutants resulting from MR were
obtained was significantly greater than was expected, suggesting that the endogenous level of MR may be under genetic control. Low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply. Human acid α-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II. Bijvoet AGA, van Hirtum H, Kroos MA, van de Kamp EHM, Schoneveld O, Visser P, Brakenhoff JPJ, Weggeman M, van Corven EJ, der Ploeg AT, Reuser AJJ: Hum Mol Genet 1999, 8:2145-2153. • Significance: A successful investigation into a potential enzyme replacement therapy for Pompe’s disease (glycogen storage disease type II). This inherited lysosomal storage disease is fatal, and there is currently no treatment. Findings: Transgenic rabbits were created using a construct containing the human acid α-glucosidase gene and a promotor to effect high expression levels in the milk of the rabbits. RhGAA (recombinant human acid α-glucosidase) was produced at levels of 8g/l milk and was purified for experiments to test its therapeutic effectiveness. Short-term, single-dose experiments in mice deficient for acid α-glucosidase showed full correction of the deficiency in all tissues except the brain. Longer-term experiments, using weekly infusions over a 6 month period resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle together with substantial improvements in tissue morphology. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients. Crooked tail (Cd) models human folate-responsive neural tube defects. Carter M, Ulrich S, Oofuji Y, Williams DA, Ross ME: Hum Mol Genet 1999, 8:2199-2204. • Significance: The genetic animal model used in this study provides a new opportunity to identify the biochemical, genetic and gender-dependent factors contributing to folate-responsive neural-tube defects. Findings: The authors produced intraspecies backcrosses of the Crooked tail (cd) mouse for linkage analysis. cd is a strain that is prone to exencephaly the most common neural tube defect in mice and rats. They mapped cd to a 0.2 cM interval of mouse chromosome 6 and were able to identify cd homozygotes on the basis of genotypes at four closely linked markers. The homozygotes were used to assess the prenatal effects of folic acid supplementation: the number of embryos affected by resorption, early lethality, and exencephaly decreased from 59%→30% when the folic acid supplementation was increased from 0→30 mg/kilogram food. It was also observed that the penetrance of cd depends on the genetic background of the mouse, implying the existence of genetic-modulating factors. Leukaemia disease genes: large-scale cloning and pathway predictions. Li J, Shen H, Himmel KL, Dupuy AJ, Largaespada DA, Nakamura T, Shaughnessy Jr JD, Jenkins NA, Copeland NG: Nat Genet 1999, 23:348-353. •• Significance: A powerful method for the identification of candidate disease genes at increased throughput, using the mouse. The authors demonstrate the effectiveness of the method in leukaemia and identify a number of genes which implicate new disease pathways. Findings: It has previously been noted that retroviral insertion mutagenesis of specific recombinant inbred mice induces a high incidence of leukaemia through activation of oncogenes or disruption of the function of tumour suppressor genes. The
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authors used these mice, and a method based on inverse PCR of proviral genomic sequences, to clone and characterize 419 integration sites. They identified all of the loci previously classified as common integration sites but also revealed a number of new common integration sites which affected loci encoding genes previously implicated in other cancers (e.g. Hras1). In addition, a number of single integration sites were identified in either loci encoding for genes with plausible roles in cancer pathways (e.g. transcription factors, kinases, phosphatases; or in genes which suggested new pathways in the pathogenesis of leukaemia, e.g. cholesterol/lipid metabolism).
mutants. Sato A, Kojima T, Ui-Tei K, Miyata Y, Saigo K: Development 1999, 126:4421-4430. • Significance: Identifies a third member of the frizzled family of wnt receptors in Drospohila and suggests that the gene product may function to attenuate wingless (wg) signaling. Findings: Dfrizzled-3 (Dfz3) expression is similar to and dependent upon wg expression in imaginal discs. Dfz3 binds wg but is not efficient at transducing the wg signal. Hypomorphic alleles of wg revert to near wild-type in the absence of Dfz3, leading to the interpretation that Dfz3 may function as a negative regulator of wg signaling.
Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium: Nature 1999, 401:921-923.
Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node. Levin M, Mercola M: Development 1999, 126:4703-4714. • Significance: The authors suggest that cellular communication between the left and right halves of early chick embryos, via gap junctions, is essential for communicating left–right pattern to Hensen’s node. Findings: Removing either left or right lateral tissues from early stage 2 chick embryos results in abnormal expression of shh and chick nodal, markers normally expressed on the left side. Inhibiting gap junction communication either pharmacologically with lindane, with antisense oligonucleotides, or with antibodies to connexin43 also disrupts normal left–right gene expression. Overall, the results demonstrate a need for cellular communication throughout the early embryo to establish a proper left–right axis.
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Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region. Shiina T, Tamiya G, Oka A, Takishima N, Yamagata T, Kikkawa E, Iwata K, Tomizawa M, Okuaki N, Kuwano Y et al.: Proc Natl Acad Sci USA 1999, 96:13282-13287. •• Significance: The nucleotide sequence of the human MHC region will facilitate positional cloning approaches aimed at defining the molecular basis of HLA-associated diseases. It also provides a new tool for studying the genetics, biology and evolutionary driving forces of human multigene families. Findings: 3.6 Mb of nucleotide sequence representing the human MHC region has been assembled from the work of 4 main groups (The MHC Consortium). Analysis of the sequence revealed the presence of at least 224 genes, of which ∼40% are estimated to have immune function. The paper by Shiina et al. details the sequencing study of a contribution to this whole MHC region, the HLA class I region. Detailed analysis of this region (1.8 Mb of sequence) showed evidence for evolution through repeated segmental duplication and subsequent diversification of a minimal building block. It is also suggested that the non-essential genes, such as HLA-F and MICE have acted as progenitors to today’s immune-competent HLA-A, B, C and MICA/B (evolution by ‘birth and death’).
Pattern formation and developmental mechanisms Selected by Mike Jones Chester Beatty Laboratories, London, UK
β and Lim1 interact in the visceral endoderm to reguHNF3β late primitive streak formation and anterior-posterior polarity in the mouse embryo. Perea-Gomez A, Shawlot W, Sasaki H, Behringer RR, Ang SL: Development 1999, 126:4499-4511. • Significance: Provides evidence that anterior–posterior and dorsal–ventral patterning of the early mouse embryo requires the synergistic functions of HNF3β and Lim1. Findings: HNF3β/Lim1 double mutant embryos exhibit enlarged primitive streaks, which leads to ectopic mesoderm formation. Dorsal mesoderm derivatives are absent, suggesting a defect in mesodermal patterning. Cer-1 and Hex1, markers of the anterior visceral endoderm are absent in double mutants as is the anterior primitive streak marker gsc. Chimeric embryo analysis demonstrates that the function of the two gene products is required in the visceral endoderm. Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic
Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system. Oppenheimer DI, MacNicol AM, Patel NH: Curr Biol 1999, 9:1288-1296. • Significance: A description of the use of a baculovirus-based misexpression system that can be used in species as diverse as insects and amphibia and its application in studying evolutionarily conserved signaling systems. Findings: Recombinant baculoviruses were used to misexpress genes in Drosophila, Tribolium, and Xenopus. Efficient infection and ectopic gene expression was observed in all species. The system was used to demonstrate that the interaction between wingless and engrailed was evolutionarily conserved between organisms as diverse as Drosophila and Tribolium. Polarity of the mouse embryo is anticipated before implantation. Weber RJ, Pedersen RA, Wianny F, Evans MJ, Zernicka-Goetz M: Development 1999, 126:5591-5598. •• Significance: This report provides the first evidence that the polarity of the postimplantation mouse embryo is related to events that occur at least as early as blastocyst stages, before implantation. Findings: Single blastocyst cells were injected with RNA encoding green fluorescent protein (GFP) to serve as a lineage tracer. Cells populating the epiblast became randomly distributed, but those colonising the visceral endoderm (VE) were distributed non-randomly. GFP-positive VE cells originally located near the polar body — a marker of the blastocyst’s axis of symmetry — came to occupy a distal portion of the egg cylinder whereas cells initially located away from the polar body generally took up a proximal (extraembryonic) position. The transformation of the blastocyst axis into the anterior–posterior axis of the embryo is suggested to begin before 5.5 days of gestation.
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Differentiation and gene regulation Selected by Alison Schuldt Wellcome/CRC Institute, Cambridge, UK
Novel functions of nanos in downregulating mitosis and transcription during the development of the Drosophila germline. Deshpande G, Calhoun G, Yanowitz JL, Schedl PD: Cell 1999, 99:271-281. •• Significance: Nanos acts through Sxl to regulate the cell cycle in germ cells and mediate their migration. Nanos additionally acts to repress transcription of several zygotic genes. Findings: Germ cells are distinct from somatic cells in that they are transcriptionally quiescent and have an attenuated cellcycle. Embryos lacking nanos transcribe certain zygotic genes in germ cells. The cell-cycle length of these cells is reduced. Extra germ cells form, which have migration defects, consistent with the proposed role for nanos in germ cell migration. Sxl is necessary and sufficient for these cell cycle and migration defects. Other targets of nanos are proposed to regulate the transcriptional repression of zygotic genes. Differential localisation of two histidine kinases controlling bacterial cell differentiation. Wheeler RT, Shapiro L: Mol Cell 1999, 4:683-694. •• Significance: The asymmetric localisation of the histidine kinases PleC and DivJ is co-ordinated with the cell cycle, demonstrating a novel mechanism for regulating polar differentiation of Caulobacter crescentus. Findings: Green fluorescent protein fusions to PleC and DivJ are followed in living cells and show that these histidine kinases are asymmetrically localised during distinct stages of the C. crescentus cell cycle. They find that PleC is required to mediate the asymmetric localisation of DivJ. This, in turn controls the phosphorylation of DivK — an essential co-ordinating factor in polar differentiation. p27Xic1, a CDK inhibitor, promotes the determination of glial cells in Xenopus retina. Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA: Cell 1999, 99:499-510. •• Significance: The CDK inhibitor p27Xic1 acts with the Notch pathway to induce the Müller glial cell fate, showing that cell cycle plays an active role in cellular determination in the retina. Findings: Overexpression of the CDK inhibitor, p27Xic1 blocks proliferation of all cell types in the devolping retina. It also leads to early differentiation of Müller glial cell type at the expense of neural cell types. Conversely, reduction of p27Xic1 function leads to a loss of Müller cells. The cyclin-binding region of p27Xic1 is responsible for this induction. Cotransfection experiments show that p27Xic1 acts as a determinant in the context of the Notch pathway. Interplay of Notch and FGF signalling restricts cell fate and MAPK activation in the Drosophila trachea. Ikeya T, Hayashi S: Development 1999, 126:4455-4463. • Significance: Notch signalling acts to restrict the fusion cell fate in trachea. It is activated by localised fibroblast growth factor (FGF) signalling between adjacent tissue and trachea through upregulation of Delta. Findings: During tracheal development, a single cell differentiates into a fusion cell at branch tips. Localised FGF signalling between exogenous tissue and trachea stimulates high expression of the Notch receptor, Delta in a single tip cell. Delta, in turn, stimulates Notch signalling in adjacent tip cells while repressing Notch autonomously. Repression of Notch in the
single Delta-expressing cell relieves inhibition of escargot and MAPK, allowing the fusion cell fate to be adopted. Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. Chen CM, Struhl G: Development 1999, 126:5441-5452. • Significance: Frizzled (Fz) and Fz2 function redundantly as the primary receptors for Wingless transduction in Drosophila. Findings: Wingless signalling is lost in the wing imaginal discs of embryos lacking both fz and fz2. Loss of both receptors leads to a loss of Armadillo, leading to the proposal that Fz and Fz2 transduce Wingless signalling through upregulation of Armadillo. Each of these is alone sufficient to confer normal Wingless signalling through development, suggesting that they are functionally redundant. nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Subramaniam K, Seydoux G: Development 1999, 126:4861-4871. • Significance: Two homologs of Drosophila nanos are required for correct differentiation of the primordial germ cells in Caenorhabditis elegans, showing that the molecular mechanisms underlying early germ-cell development is evolutionarily conserved. Findings: In this study, three nanos-related genes — nos-1, nos-2, and nos-3 — are identified. nos-1 and nos-2 are expressed in germ cells and function redundantly during later germline development. nos-1 and nos-2 are required to both maintain germ cell viability post-embryonically and control germ-cell proliferation. RNA-interference experiments suggest that nos-1 and nos-2 may function with a RNA-binding protein related to Pumilio in Drosophila. Selected by Massimiliano Andreazzoli National Institute of Child Health and Human Development, Bethesda, Maryland, USA
Oxygen regulation of airway branching in Drosophila is mediated by Branchless FGF. Jarecki J, Johnson E, Krasnow MA: Cell 1999, 99:211-220. •• Significance: The Drosophila respiratory system consists of primary, secondary and terminal branches. The pattern of terminal branches is remarkably variable, unlike the pattern of primary and secondary branches which is rigidly established. This paper shows that Branchless, (Bnl; Drosophila fibroblast growth factor) acting as a local signal generated by oxygenstarved cells is necessary and sufficient for terminal branching. Thus, Bnl, being also involved in primary and secondary branches specification, appears to be used repeatedly at different levels of trachael branching. Findings: Oxygen deprivation was found to stimulate both Bnl expression and terminal branch sprouting. Ectopic expression experiments demonstrated that Bnl induces terminal branch proliferation whereas expression of a dominant negative form of Bnl receptor was shown to block terminal branching. Mice lacking both presenilin genes exhibit early embryonic patterning defects. Donoviel DB, Hadjantonakis AK, Ikeda M, Zheng H, Hyslop PS, Bernstein A: Genes Dev 1999, 13:28012810. • Significance: Presenilins are cytoplasmic proteins which have been implicated in the positive regulation of the Notch signalling pathway. Combinatorial inactivation of the two murine
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presenilins, PS1 and PS2, demonstrates that these proteins play different but partially overlapping roles in many developmental processes. Findings: Whereas PS1 null mice die perinatally because of several developmental defects, PS2 null mice or PS2 null/PS1 heterozygous mice are normal. Inactivation of one or both copies of PS2 in PS1 null mice, however, gives rise to increasingly severe and pleiotropic defects which might be the consequence of an abnormal mesoderm patterning. Although some aspects of the double null phenotype are similar to the phenotype of mice lacking RBP-Jk, a transcription factor activated by Notch, the widespread defects observed in presenillin null embryos suggest that these proteins might also function outside the Notch pathway. Targeted mutagenesis of the endogenous mouse Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Arango NA, Lovell-Badge R, Behringer RR: Cell 1999, 99:409-419. • Significance: Müllerian inhibiting substance (MIS), a protein secreted by Sertoli cells derived from fetal testis, is essential to cause regression of Mullerian ducts in mammalian males. This work demonstrates the importance of steroidogenic factor 1 (SF1) and SOX9 (a high mobility group protein) binding sites, present in Mis promoter, for gene activity in vivo. Findings: Mutations in SF1 and SOX9 binding sites were introduced by homologous recombination. Whereas mutations in the SF1 site cause a reduction of Mis expression which does not substantially affect sexual development, mutations in the SOX9 site abolish Mis expression resulting in complete retention of Müllerian ducts in male mice. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K, Benezra R: Nature 1999, 401:670-677. •• Significance: Proteins which inhibit DNA binding and cell differentiation (Id proteins) are known to control cell differentiation by heterodimerizing with basic helix-loop-helix (bHLH) transcription factors and thus interfering with their DNA-binding activity. Here, it is shown that Id proteins are required for the correct timing of neuronal differentiation and to support angiogenesis during normal development and in tumour xenografts. Findings: Mice lacking both Id1 and Id3 display a precocious and ectopic expression of neural bHLH genes and an absence of the normal branching and sprouting of capillaries in the neuroectoderm. Intradermal injection of tumour cell lines in mice lacking Id3 and only one copy of Id1 showed a remarkable resistance of these mice to tumour growth as a result of a lack of vascularization of the xenograft. Mesenchymal to epithelial conversion in rat metanephros is induced by LIF. Barasch J, Yang J, Ware CB, Taga T, Yoshida K, Erdjument-Bromage H, Tempst P, Parravicini E, Malach S, Aranoff T, Oliver JA: Cell 1999, 99: 377-386. •• Significance: Signals produced by the ureteric bud are necessary to convert the metanephric mesenchyme to epithelia. This work shows that these signals are mediated by the leukemia inhibitory factor (LIF) and other cytokines of the IL-6 family. Findings: Purification of the epithelializing activity of media conditioned by ureteric bud cells led to the isolation of LIF. A transient exposure to LIF is sufficient to trigger epithelialization of metanephric mesenchyme and subsequent formation of renal
tubules and glomeruli. LIF exerts its effects acting on a cluster of epithelial precursor expressing Pax2 and Wnt4. Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding. Porcher C, Liao EC, Fujiwara Y, Zon LI, Orkin SH: Development 1999, 126:4603-4615. •• Significance: Basic helix-loop-helix (bHLH) transcription factors are thought to regulate cell differentiation through their DNA binding activity. This work demonstrates that stem cell leukemia (SCL), a bHLH factor involved in hematopoiesis and vascular development, is able to exert most of its functions in vivo in a DNA-binding-independent manner. Findings: To define regions of SCL essential for its function, variant forms of the protein were tested for their ability to rescue murine ES cells lacking SCL and the vascular and hematopoietic defects of the zebrafish mutant cloche. The DNA-binding activity of SCL is found to be dispensable to rescue primitive erytropoiesis and to initiate hematopoiesis and vascular development. SCL DNA binding activity is required for the generation of the correct number of erythroid precursor and for erythroid and megakaryocytic precursor maturation.
Genomes and evolution Selected by Susan Douglas Institute for Marine Bioscience, Halifax, Nova Scotia, Canada
A pair of back-to-back papers in Nature describing the sequences of major histocompatibility loci from chicken and human show the dramatic differences in evolutionary strategies employed in the common fight against disease by two groups that diverged from one another 300 million years ago. Complete sequence and gene map of a human major histocompatibility locus. The MHC sequencing consortium: Nature 1999, 401:921-923. •• Significance: A major collaboration from four sequencing groups has revealed 224 gene loci comprising the three classes of mammalian major histocompatability complex (MHC) genes in a 3.6 Mb region of human chromosome 6. As many of these genes are involved in the immune response, this information opens the door to a better understanding of these highly polymorphic genes, their multigene families and the identification of disease loci. Findings: The region of chromosome 6 presented in this study includes the MHC genes in the ‘classical’ Class I, II and III regions as well as the immediate flanking regions which contain additional genes referred to as ‘extended’ Class I and II genes. Immune-related genes comprise ∼40% of the total and are clustered in the ‘classical’ Class II region. Gene density is 1 per 16 kb and pseudogenes abound, particularly in Class I and II regions. Gene duplications have also occurred frequently in these two regions, resulting in new gene family members with distinct functions. The 2% rate of meiotic recombination between the widely-distributed polymorphic genes has resulted in a huge variety of haplotypes in humans. The chicken B locus is a minimal essential major histocompatibility locus. Kaufman J, Milne S, Göbel TWF, Walker BA, Jacob JP, Auffray C, Zoorob R, Beck S: Nature 1999, 401:923-925. •• Significance: Sequence analysis of the 19 genes present in a 92 kb region of the BF/BL region of the chicken B locus
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shows both striking similarities and remarkable contrasts to its human MHC counterpart. The chicken MHC exhibits a strong association with resistance and susceptibility to certain infectious pathogens, and thus provides a simple model for investigating the molecular basis for MHC-determined pathogen-specific disease resistance. Findings: Although most of the genes in the chicken locus have counterparts in the human MHC gene classes, they are organized differently and much more compactly — short introns and intergenic regions, few repeats, no pseudogenes. Furthermore, genes expected to be present (e.g. proteasome components) are absent and genes not found in human MHC (e.g. natural killer [NK] receptor, are present). The compressed structure of the chicken MHC allows co-evolution of genes resulting in haplotypes that are relatively stable over evolutionary time.
The DNA sequence of human chromosome 22. Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Brunswick R, Beare DM, Clamp M, Smink LJ et al.: Nature 1999, 402:489-495. •• Significance: This landmark paper from the Human Genome Project (HGP) presents the first glimpse of a complete human chromosome and provides insights into the information to be gleaned from the remaining chromosomes. Chromosome 22 is the second smallest autosomal chromosome and is one of five human acrocentric chromosomes. Its gene content is rich and it contains many regions associated with human disease including schizophrenia and spinocerebellar ataxia. The sequence provides a model for the testing of computational tools for gene prediction and repeat element recognition. (See also alert by Dawson, p5.) Findings: The 33.4 Mb of sequence was obtained using the HGP ‘clone by clone’ approach and is presented as 12 contiguous segments separated by gaps of <150 kb that were intractable to sequencing using available cloning systems. The 545 annotated genes and 134 pseudogenes comprise 39% of the total sequence and exhibit a wide range in size from 1 kb to >583 kb. Tandem and interspersed repeat sequences comprise 41.9% of the genome. A number of duplicated gene families and low-copy repeats have been identified. Physical locations of microsatellite markers have been assigned to the chromosome and regions differing dramatically in G+C content (isochores) have been mapped. Comparison with the mouse genome reveals 8 regions of conserved synteny and allows sites of evolutionary rearrangements to be identified accurately at the nucleotide level.
Novel coding regions in four complete archaeal genomes. Raghavan S, Ouzounis CA: Nucleic Acids Res 1999, 27:4405-4408. • Significance: Prediction of coding sequences from genomes is not always perfect as a result of incomplete annotation, sequencing errors and the limitations in functional information of many genes. Understandably, many ‘non-coding’ regions may therefore actually contain potential protein-coding regions that have escaped detection. This paper compares all of the regions between annotated open reading frames (ORFs) in each of four completely sequenced archaeal genomes in an attempt to identify potential coding regions. Findings: 2237 inter-ORF regions from the complete genomes of Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus and Pyrococcus horikoshii were used to query the non-redundant protein sequence database using BlastX v. 2.0. 524 regions contained complete ORFs (113) and sections of previously characterised ORFs with conflicting start/end sites (411). The closest homologs of most of the newly identified ORFs were other archaeal proteins. In 21 cases, the ORF had similarity to a protein of known function. This finding underscores the importance of regularly matching ‘non-coding regions’ against protein databases. The distribution of RNA motifs in natural sequences. Bourdeau V, Ferbeyre G, Pageau M, Paquin B, Cedergren R: Nucleic Acids Res 1999, 27:4457-4467. • Significance: The identification of RNA genes and structures in sequenced genomes has not been given the attention that their protein-coding counterparts have. This paper describes the search for such RNA structures using motifs and introduces the concept of ‘ribonomics’. The motifs are broadly categorized as protein-binding, chemically and catalytically active, and small molecule-binding. The origin of motifs and the significance of their wide distribution in the database can now be discussed and the knowledge used in logical RNA based-intervention for modulating biological processes. Findings: Using the computer search engine RNAMOT, searches of both strands of sequence data in GenBank as well as a random sequence database of 10,000 sequences of 100,000 nucleotides each were carried out. The origin of the majority of motifs by random evolutionary drift is supported by comparison of the distribution of motifs in natural sequences versus random sequences. An RNA motif database comprising the distribution of motifs among 67 GenBank features as well as their phylogenetic distribution was constructed and is available at http://www.centrcn.umontreal.ca/bourdeav/Ribonomics