Genetics and development

gda2pap.qxd

03/22/2000

02:34

Page 129

129

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, 10:129–137 Contents (chosen by) 129 Oncogenes and cell proliferation (Mittnacht and Roche) 131 Chromosomes and expression mechanisms (Aasland and

Weinzierl) 132 134 135 136 • ••

Genetics of disease (Dawson) Pattern formation and developmental mechanisms (Jones) Differentiation and gene regulation (Schuldt and Tsang et al.) Genomes and evolution (Douglas) of special interest of outstanding interest

Oncogenes and cell proliferation Selected by Sibylle Mittnacht Institute of Cancer Research, Chester Beatty Laboratories, London, UK

Regulation of CDK4 activity by a novel CDK4-binding protein, p34 SEI-1. Sugimoto M, Nakamura T, Ohtani N, Hampson L, Hampson IN, Shiamoto A, Furuichi Y, Okumura K, Niwa S, Taya Y, Hara E: Genes Dev 1999, 13: 3027-3033. •• Significance: The report describes a novel protein that can facilitate activation of CDK4 by cyclin Ds in the presence of INK4a. This finding provides an important lead as to how cells may manage to generate active Cyclin D/cyclin-dependent kinase (CDK) complexes in the face of inhibitory levels of InK4a protein. Findings: Using INK4a as a bait in a yeast interaction screen, Sugimoto et al. identify a novel protein, p34 SEI-1 (selected with INK4A). In vitro p34 SEI-1 specifically interacts with CDK4 (but not CDK6), facilitates formation of a complex containing CDK4/INK4a and cyclin D and renders cyclin D/CDK4 complexes resistant to INK4a inhibition. In cells, p34 SEI-1 is induced by serum and, upon ectopic expression, triggers proliferation and the formation of active cyclin D/CDK4 complexes in the absence of growth factor. Oppositely imprinted genes p57KIP2 and IGF2 interact in a mouse model for Beckwith-Wiedemann syndrome. Caspary T, Cleary MA, Perlman EJ, Zhang P, Elledge SJ, Tilghman SM: Genes and Dev 1999, 13:3115-3124. •• Significance: Maternal loss-of-function of the cyclin-dependent kinase inhibitor p57KIP2 or increased expression of insulin like growth factor-2 (IGF-2) have been implicated independently in the development of Beckwith–Wiedemann syndrome (BWS) in man. Mice with either a maternal p57KIP2 defect or IGF-2 overexpression, however, show only an incomplete, partially overlapping subset of phenotypes classically associated with BWS. The above report demonstrates that a near complete BWS phenotype is developed in mice in which loss of p57KIP2 is combined with loss of IGF-2 imprinting. This suggests for the first time that lack of p57KIP2 function may

cooperate with increased IGF-2 expression towards development of the syndrome. Findings: BWS is characterised by malformation and somatic overgrowth in various organs as well as increased susceptibility to childhood cancers. The authors describe the generation of a mouse model in which a null mutation of p57KIP2 is combined with loss-of-imprinting of IGF-2 (a mutation leading to overproduction of IGF-2 in organs with IGF-2 imprinting). Mice with such combined defects show a near full set of BWS-associated abnormalities. Interestingly, interbreeding of double-defective females with males carrying a defective IGF-2 allele results in triple mutant embryos which show rescue of those defects normally associated with this defective IGF-2 allele, this suggesting that p57KIP2 loss may compensate functionally for decreased IGF-2 signalling. Direct inhibition of G1 cdk kinase activity by MyoD promotes myoblast cell cycle withdrawal and terminal differentiation. Zhang J-M, Zaho X, Wei Q, Paterson BM: EMBO J 1999, 18:6983-6993. • Significance: Previous results indicated that muscle differentiation is impaired in cells lacking pRB or the pRB-like pocket-protein p107 and suggested that Myo D may be a downstream target for these pocket proteins. This report suggests that MyoD can act upstream of the pocket proteins, causing pocket protein activation and consequential cell-cycle exit by inhibiting Cyclin D activated kinases. Findings: The authors report that a 15 residue long amino acid stretch in the carboxy-terminal portion of MyoD binds to the catalytic cyclin-dependent kinase subunit, CDK4, and can inhibit CDK4/cyclin D complexes in vitro. Expression of this amino acid stretch in myoblast cells triggers cell-cycle exit and myogenic differentiation in full serum (i.e. conditions under which differentiation does not normally occur). Furthermore, this stretch is necessary, but not sufficient, for the induction of muscle differentiation in non-MyoD expressing cells. Regulation of tumour angiogenesis by p53-induced degradation of hypoxia-inducible factor. Ravi R, Mookerjee B, Bhujwalla ZM, Sutter CH, Artemov D, Zeng Q, Dillehay LE, Madan A, Semenza GL, Bedi A: Genes Dev 2000, 14:34-44. •• Significance: The study indicates that inactivation of the p53 tumour suppressor protein in cancer cells provides a potent stimulus for tumour angiogenesis. Findings: The authors use isogenic tumour cells that only differ in the expression of p53 to demonstrate that p53 loss greatly stimulates neovascularization and tumour forming ability in mice, results in increased VEGF (vascular endothelial growth factor) mRNA expression and stabilisation of HIF (hypoxiainduced factor) — itself an important regulator of the VEGF gene. They furthermore provide evidence that functional p53 interacts directly with HIF-1α, targeting it for proteasome-mediated degradation via ubiquitination involving Hdm2 (human double minute 2) ubiquitin ligase. A genome-wide survey of RAS transformation targets. Zuber J, Tchernitsa OI, Hinzmann B, Schmitz A-C, Grips M,

gda2pap.qxd

03/22/2000

130

02:34

Page 130

Paper alert

Hellriegel M, Sers C, Rosenthal A, Schaefer R: Nat Genet 2000, 24:144-152. • Significance: The report provides a catalogue of some 400 genes differentially expressed in RAS-transformed cells. Along with other types of tumour profiling, such information should be useful for narrowing down the genes which are significant for executing the repertoire of biological changes in tumourigenic cells. Findings: By using subtractive suppression hybridisation, the authors identify genes expressed differentially in isogenic rat cells that differ in the expression of oncogenic V12-RAS (HRAS). They identified 104 expressed sequence tags, 45 novel sequences and 244 known genes that were either downor-upregulated in the presence of HRAS. Intriguingly, only a subset of these genes appears to be regulated upon transformation by other members of the RAS family (i.e. KRAS and NRAS) and only a rather small portion (61 genes) seem directly regulated by the RAS effector pathway involving MEK. Selected by Serge Roche Centre National de la Recherche Scientifique, Montpellier, France

Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Miao H, Burnett E, Kinch M, Simon E, Wang B: Nat Cell Biol 2000. 2:62-69. • Significance: Receptors of the Eph family are tyrosine kinases with important functions in cellular guidance but their modes of intracellular signalling are poorly defined. The authors here identify an intracellular molecular mechanism by which Eph2A promotes repulsive guidance of cell migration. Findings: Eph2A (also called Eck) is found to be the main receptor of the Eph family expressed in the prostate epithelial cell line PC-3. Activation of the receptor induced transient cell rounding, inhibited integrin-mediated cell adhesion, cell spreading and migration. Eph2A maintained integrins in an inactivate conformation, the cytoplasmic focal adhesion tyrosine kinase Fak was associated with the receptor in resting cells, and its activity was down-regulated upon cell-stimulation. Finally, evidence for an involvement of the tyrosine phosphatase Shp2 is also provided: Shp2 became rapidly tyrosine phosphorylated upon Eph2A activation, dephosphorylated Fak in vivo and dominant negative Shp2 substantially reversed receptor-induced cell-shape rounding. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Tanoue T, Adachi M, Moriguchi T, Nishida E. Nat Cell Biol 2000, 2:110-116. • Significance: Mitogen activated protein kinases (MAPKs) are found to interact with their activators, modulators and substrates through a common docking sequence. This mechanism may have important implications on the regulation of the MAPK signalling cascade. Findings: A cluster of positive charged amino acids was identified in the activators (MAP kinase kinase: MEKs), regulators (MAPK phosphatases: MKPs), and substrates (MAPK activated protein kinases: MNKs) of the MAPK for efficient binding. Mutagenesis of this region reduced binding of the activator MEK1, the regulator MKP3 and the substrate MNK1 to the MAPK ERK2. A short conserved sequence composed of negatively charged residues was subsequently identified in MAPK for docking (called common domain or CD). This sequence lies outside the kinase domain and close to the sequence involved in protein dimerization. Although the CD was not involved in

kinase activity per se, it increased efficiently enzymatic reactions. Finally a similar mechanism was observed for p38 and Jun amino-amino terminal kinases. Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinases. Tominaga T, Sahai E, Chardin P, McCormick F, Courtneidges SA, Alberts AS: Mol Cell 2000, 5:13-25. •• Significance: The small GTP binding protein Rho is an important regulator of the actin cytoskeleton, cell division, and expression of serum response factors (SRFs) but the Rhomediated pathways are ill defined. The authors here identify the cytoplasmic tyrosine kinase Src as an important down-stream element of the Rho-effector diaphenous-related formin protein. Findings: Mouse related diaphanous proteins mDia1 and mDia2 are two effectors of Rho with formin homology (FH). Src was found to be a target of mDia: mDia associates with SrcSH3, and active Src colocalizes with mDia. Functional relevance of this interaction was assessed by microinjection approaches: in addition to G2 progression, Src regulates cytokinesis and this effect also implicates mDia. Active mDia also promotes actin stress fibres and SRF activation in a Srcdependent manner. Regulation of JNK by Src during Drosophila development. Tateno M, Nishida Y, Adachi-Yamada T: Science 2000 287:324-327. • Significance: Physiological evidence for the Jun amino-terminal kinase (JNK) as an effector for Src is now confirmed in Drosophila. Findings: the Drosophila JNK Bsk regulates both dorsal closure of the epidermis in embryos and thorax closure in pupaes. In the search for upstream regulators of the JNK pathway, the authors identify Src42A, a drosophila member of the Src family. Strong mutants for Src42A induced a phenotype that resembles Tec29, a gene encoding a cytoplasmic tyrosine kinase of the Tec family, previously identified as an effector for Src64. This confirms redundancy between Src members and suggests an involvement of Tec in the regulation of the JNK pathway. Indeed the Tec29 Src42A double mutant induced a lethal phenotype that was partially rescued by expression of activated DJun. Finally, forced expression of an activated Src42A induced phosphorylation of Bsk in agreement with a regulation JNK by Src at the post-translational level. Regulation of carbamoyl phosphate synthetase by MAP kinase. Graves LM, Guy HI, Kozlowski P, Huang M, Lazarowski E, Pope RM, Collins MA, Dahlstrand EN Ill, Evans DR: Nature 2000, 403:328-330. • Significance: Increase in nucleotide level is required for DNA synthesis and gene expression for protein synthesis, two important events of cell growth. An unexpected link between uridine nucleotide synthesis and mitogen-activated protein (MAP) kinase has been now established. Findings: carbamoyl phosphate synthetase II (CPSII) activity of the CAD protein is the rate-limiting activity for de novo synthesis of uridine nucleotides in mammalian cells. CPSII activity correlates with cell growth index. In the search for substrates of MAP kinase, the CAD protein was identified and a consensus sequence for MAPK phosphorylation was subsequently found. Evidence for an involvement of MAPK in mitogen-induced CPSII activity is also provided: allosteric stimulation of CPSII activity was obtained when phosphorylated by MAP kinase in

gda2pap.qxd

03/22/2000

02:34

Page 131

Paper alert

vitro or by stimulating cells with EGF; CAD was phosphorylated in cells stimulated by EGF; finally EGF increased the Uridine 5′ trisphospate (UTP) cell-content in a MAPK-dependent fashion. Reversibility of acute B-cell leukemia induced by BRCABL1. Huettner CS, Zhang P, Van Etten A, Tenen DG: Nat Genet 2000, 24:57-60. • Significance: The oncogene BCR-ABL is required for both induction and maintenance of leukemia, confirming BCR-ABL tyrosine kinase as important target for leukemia therapy. Findings: A conditional mouse strain for BCR-ABL was generated in order to circumvent embryonic lethality. Mice developed acute pre-B cell leukemia upon BCR-ABL expression which was strongly reversed when the transgene was downregulated. Rapid disappearance of leukaemia was attributed to transformed-cell apoptosis rather than dedifferentiation. A distinct behaviour was noticed, however, in a mouse line carrying the same transgene: leukemia persisted even in the absence of BCR-ABL, suggesting that secondary mutation has occurred, causing BCR-ABL-independent disease. Negative regulation of lymphocyte activation and autoimmunity by the molecular adapter Cbl-b. Bachmaier K, Krawczyk C, Kozieradzki I, Kong YY, Sasaki T, Oliveira-dosSantos A, Mariathasan S, Bouchard D, Wakeham A, Itie A, et al.: Nature 2000, 403:211-216. AND Cbl-B regulates CD28 dependence of T-cell activation. Chiang YJ, Kole HK, Brown K, Naramura M, Fukuhara S, Hu RJ, Jang IK, Gutkind JS, Shevach E, Gu H. Nature 2000, 403:216-220. • Significance: An important function for the adapter Cblrelated protein Cbl-b in negative regulation of lymphocyte activation and auto-immunity. Findings: A Cbl-b knockout mouse is presented. Although Cblb disruption is not embryonically lethal, Cbl-b–/– mice develop spontaneous auto-immunity. Corresponding T-cells show a higher rate of proliferation and a hyper-production of their growth factor interleukin-2 (IL-2). Evidence is provided for a function of Cbl-b in CD28 activation: Cbl-b–/– T cells do not require CD28 engagement for activation, and do not secrete IL2 upon activation — response which was restored in CD28–/– Cbl-b–/– T cells. Tyrosine phosphorylation of the Rho/Rac/Cdc42 activator Vav was also enhanced in Cbl-b deficient cells, suggesting that Vav is an important target of Cbl-b. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage proHB-EGF. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. Nature 1999, 402:884-888. •• Significance: Trans-activation of the EGF receptor (EGFR) is an important component of the cellular signalling pathway induced by seven transmembrane receptors coupled to G protein. The molecular mechanism for such activation is, however, still obscure. An unexpected mechanism for trans-activation has been now discovered. Findings: Thrombin-induced trans-activation of the EGF but not the PDGF receptor in Rat1 fibroblasts. Thrombin also induced trans-activation of the chimeric receptor composed of the EGFR extracellular domain and the transmembrane and the cytoplasmic tail of the PDGFR, in favour of an EGF paracrine loop. The authors identified HB-EGF as a key player in this process: HB-EGF has a strong affinity for both EGFR and cell-

131

surface heparan sulfate which increases the local concentration of the ligand at the cell surface without any diffusion into the extracellular medium. This may explain why EGF secretion was not previously detected. HB-EGF was generated by processing of the proHB-EGF precursor via a metalloprotease. Evidence for such a protease-dependent mechanism is provided by the use of the specific inhibitor batimastat. The nature of the protease involved in this process was, however, not reported. A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK. Barila D, Mangano R, Gonfloni S, Kretzschmar J, Moro M, Bohmann D, Superti-Furga G: EMBO J 2000, 19:273-281. • Significance: The report of a newly discovered tyrosine phosphorylation circuit in the nucleus. Findings: A consensus sequence for phosphorylation of the transcription factor cJun by cAbl is identified and, indeed, overexpression of a nuclear cAbl allele induces tyrosine phosphorylation of cJun at tyrosine 170. Phosphorylated cJun associates with Abl-SH2 and stimulates tyrosine kinase activity. Abl is known to increase Jun amino-amino terminal kinase (JNK) activity and evidence is provided for an connection between cJun level, cAbl and JNK activities. The physiological significance of cJun tyrosine phosphorylation was, however, not reported.

Chromosomes and expression mechanisms Selected by Rein Aasland University of Bergen, Bergen, Norway

An early developmental transcription factor complex that is more stable on nucleosome core particles than on free DNA. Cirillo LA, Zaret KS: Mol Cell 1999, 4:961-969. • Significance: Although most transcription factors are prevented from accessing binding sites embedded in nucleosomes, some transcription factors also bind well to nucleosomal sites. A transcription factor that initiates assembly of proteins on an enhancer, however, might be expected to bind more effectively to nucleosomal than to free DNA. This property is indeed demonstrated here for hepatocyte nuclear factor 3 (HNF3), a transcription factor that binds to the albumin enhancer even before the gene becomes active. Findings: A part of the albumin enhancer that contain HNF3binding sites was assembled as a dinucleosome in vitro. Using competition experiments, it was shown that HNF3 associates more stably with the binding sites on nucleosomes than on free DNA. An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing. Tanny JC, Dowd GJ, Huang J, Hilz H, Moazed D: Cell 1999, 99:735-745. • Significance: The Sir2 protein (Sir2p) is involved in different types of gene silencing in yeast. At telomeres and the matingtype loci, Sir2p is part of a protein complex that interacts with nucleosomes and mediates formation of silent chromatin. The exact role of Sir2p in silencing ís, however, not known although Frye has recently shown that Sir2p-like proteins have ADP-ribosyltransferase activity (Biochem Biophys Res Commun 1999 260:273). The findings described in the present report suggest that Sir2p-mediated silencing involves ADP-ribosylation of nucleosomal histones. Findings: Sir2p is found to catalyse ADP-ribosylation of both itself as well as nucleosomal core histones in vitro. A mutation

gda2pap.qxd

03/22/2000

132

02:34

Page 132

Paper alert

in Sir2p that abolishes this activity was found to compromise the silencing activity of the protein in vivo. Targeted mRNA degradation by double-stranded RNA in vitro. Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA: Genes Dev 1999, 13:3191-3197. • Significance: Double-stranded RNA interference, RNAi, is a post-transcriptional gene silencing phenomenon observed in many organisms, including nematodes, flies and vertebrates. Although both ribonuclease and RNA-directed RNA polymerase activities have been implicated, the mechanism underlying RNAi remains elusive. In the present work, an in vitro system has been established that recapitulates several aspects of RNAi. This system should be useful in the further delineation of the RNAi mechanism. Findings: Targeted mRNA degradation is observed in cell-free extracts from syncytial blastoderm-stage Drosophila embryos. Most importantly, RNA degradation is only observed with double stranded RNA molecules that correspond to a part of the mRNA — a hallmark of RNAi. A distal heterochromatic block displays centromeric activity when detached from a natural centromere. Platero JS, Ahmad K, Henikoff S: Mol Cell 1999, 4:995-1004. • Significance: Faithful segregation of chromosomes at mitosis requires functional centromeres. In bakers yeast, a short and unique sequence element fulfills this role. In animal and plant centromeres, however, no such sequence elements have been identified. In these organisms, centromeres are primarily composed of large blocks of repeated sequences that form heterochromatin. Yet, all heterochromatin sequences do not function as centromeres. Data presented here support the idea that chromosomes can have several centromere-competent regions, one of which is normally dominant. Findings: Using site-specific recombination in Drosophila, the authors generated chromosome fragments that carry the brownDominant -allele, a locus that contains a large heterochromatic region. These fragments moved properly at anaphase and displayed a significant competence for stable segregation through several cell generations. Selected by Robert OJ Weinzierl Imperial College of Science, Technology and Medicine, London, UK

Aberrant CpG-island methylation has non-random and tumour-specific patterns. Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Feramisco JD, Peltomaki P, Lang JC et al.: Nat Genet 2000 24:132-138. •• Significance: The differential methylation of CpG islands in higher eukaryotic genomes has important functional implications for various nuclear processes, such as DNA replication, chromatin condensation and transcription. Expressed genes usually contain unmethylated CpG islands. This study describes a new two-dimensional gel technique that allows the large-scale scanning of the methylation status of thousands of CpG islands in a variety of primary human tumors and thus provides a powerful analytical tool for investigating the link between tumorigenesis and aberrant genomic methylation. Findings: ‘Restriction landmark genomic scanning’ (RLGS) takes advantage of the property of the restriction endonuclease Not I to cleave only target sites located in unmethylated CpG islands. After radioactive endlabelling and further enzymatic digestions the DNA fragments are run on a two-dimensional gel

to reveal a complex pattern of spots that provide a distinct fingerprint of the genome methylation status. The authors estimate that ~600 CpG islands out of 45,000 present in the human genome are aberrantly methylated in primary tumors, some in a tumor type-specific manner. Positive and negative regulation of endogenous genes by designed transcription factors. Beerli RR, Dreier B, Barbas CF III: Proc Natl Acad Sci USA 2000 97:1495-1500. • Significance: Many conventional approaches focusing on the manipulation of the expression of selected genes are based on post-transcriptional ‘antisense’ techniques. The ability to control the expression levels of particular genes with ‘designer transcription factors’ is still in its infancy but has potentially many exciting applications in biology and medicine. Findings: Artificial DNA-binding domains based on standard ‘zinc finger’ Cys2-His2 motifs were designed to bind specifically to 18 nucleotide DNA target sequences in the 5′ untranslated regions of the endogenous erbB-2 and erbB-3 genes (these protooncogenes are frequently overexpressed in human cancers). The artificial DNA-binding domains were fused by genetic engineering either to multiple copies of the VP16 activation domain or to the KRAB repressor domain and introduced into the human carcinoma cell lines A431 and HeLa. As predicted, the production of the synthetic transcription factors either increased erbB expression by almost six-fold (VP16 construct), or repressed transcription of the genes to below detection level (KRAB construct). Additional experiments proved the high degree of specificity obtainable in this system and defined some of the criteria for the optimal design of engineered DNA-binding domains. A testis-specific transcription factor IIA (TFIIAt) stimulates TATA-binding protein-DNA binding and transcription activation. Ozer J, Moore PA, Lieberman PM: J Biol Chem 2000, 275:122-128. • Significance: General transcription factors, such as transcription-factor IIA (TFIIA), are considered to form part of a largely invariant basal transcriptional machinery that is programmed by transcription factors which are expressed in a celland tissue-specific manner. This paper identifies a human ‘TFIIA-related protein’ (TFIIAt) that is predominantly expressed in testis tissues and displays functional properties similar to ubiquitously expressed TFIIA. Findings: TFIIA binds directly to the TATA-binding protein (TBP) and has a generally stimulatory effect on the transcription of many promoters. The testis-specific version, TFIIAτ, forms complexes with TBP indistinguishable from those formed by TFIIA and supports activated transcription in the presence of most activators in TFIIA-depleted nuclear extracts. TFIIAτ does not, however, support activated transcription by Zta (an Epstein-Barr virus encoded transcription factor), thus suggesting that TFIIAτ displays at least some specialised tissue-specific functions.

Genetics of disease Selected by Elisabeth Dawson The Sanger Centre, Cambridge, UK

A human model for multigenic inheritance: Phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus. Bolk S, Pelet A, Hofstra RMW, Angrist M, Salomon R, Croaker D, Buys CHCM, Lyonnet S, Chakravarti A: Proc Natl Acad Sci USA 2000, 97:268-273.

gda2pap.qxd

03/22/2000

02:34

Page 133

Paper alert

•• Significance: This study provides evidence for the existence of multiple genes contributing to a multigenic disease. It illustrates how the identification of new susceptibility factors for a complex disease can be enhanced when the families being used can be classified according to mutation type at known susceptibility genes. Specifically, the nature of the RET mutation in Hirschprung disease (HSCR) patients is a strong predictor of the presence (segregation) of additional genetic changes, supporting multigenic inheritance. Findings: HSCR is a complex genetic disorder and coding sequence mutations in the receptor tyrosine kinase RET can be identified in ~50% of familial cases. In this investigation of 12 multiplex families with HSCR, 11 were linked to the RET locus on chromosome 10. Mutation analysis of the coding region of the RET gene showed that 6 of these families harbored mutations at an evolutionarily conserved amino acid; mutations that are likely to represent loss of function variants. A genome scan using all 12 families revealed another linked locus on chromosome 9q31. The segregation of the 9q31 locus was restricted to the families that do not have missense or nonsense RET mutations.

133

as a tumour suppressor gene conferring predisposition to sarcoma, breast cancer and brain tumours. Findings: TP53 mutations account for 75% of LFS cases. This study focussed on four LFS families that did not have TP53 mutations. The authors looked for mutations in the human counterparts of genes previously identified in yeast as playing a major role in G2 checkpoint control (kinases that are activated in repsonse to DNA damage and thus prevent cellular entry into mitosis). In one of these families, three LFS patients had an identical mutation (1100delC) in one copy of the gene encoding hCHK2. The mutation was not evident in a healthy relative or 50 controls. The authors looked at this gene in 18 patients suffering from a related syndrome called variant LFS and in 49 cancer cell lines from a variety of non-hereditary tumours. hCHK2 was mutated in three cases and not at all in 50 healthy volunteers. Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Cao H, Hegele RA: Hum Mole Genet 2000, 9:109-112. AND

Large-scale test of hypothesised associations between the angiotensin-converting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5000 cases and 6000 controls. Keavney B, McKenzie C, Parish S, Palmer A, Clark S, Youngman L, Delépine M, Lathrop M, Peto R, Collins R, for the International Studies of Infarct Survival (ISIS) Collaborators: The Lancet 2000, 355:434–442. • Significance: This study highlights the need for some association studies of candidate genes and complex diseases to involve much larger populations than is customary, and to have less emphasis on retrospectively defined subgroups. Findings: Angiotensin-I-converting enzyme (ACE) has been proposed as a candidate for susceptibility to cardiovascular disease in view of its physiological role and the established benefits of ACE-inhibitor therapy. This International Studies of Infarct Survival (ISIS) study aimed to clarify the conflicting results from previous association studies of an insertion/deletion (I/D) polymorphism of the ACE gene and myocardial infarction (MI). There have some suggestions that the DD genotype confers increased susceptibility to MI. In this study, the authors looked at the frequencies of the insertion/deletion polymorphism in the angiotensin-I-converting enzyme (ACE) in 4629 myocardial infarction cases and 5934 controls. The ACE DD genotype was found in 1359 (29.4%) of the MI cases and in 1637 (27.6%) of the controls; a weak but non-significant association. The patients were stratified into subbgroups dependent on their risk of MI; plasma apolipoprotein B concentration, genotype at the angiotensin-II type-1 receptor gene, sex, and age; an exploratory analysis showed that each subgroup had similar frequency of the DD genotype.

LMNA, encoding lamin A/C, is mutated in partial lipodystrophy. Shackleton S, Lloyd DJ, Jackson NJ, Evans R, Niermeijer MF, Singh BM, Schmidt H, Brabant G, Kumar S, Durrington PN et al.: Nat Genet 2000, 24:153-156. •• Significance: These are the first reports of a gene — nuclear lamin A/C (LMNA) — found to be altered in a degenerative disorder of adipose tissue. They suggest that LMNA mutation could underlie other diseases characterized by tissue type- and anatomical site-specific degeneration and that other proteins and function of the nuclear envelope may be pathological in adipose tissue and insulin action. Findings: Partial lipodystrophy (PLD) is characterised by normal fat distribution until after puberty, when regional and progressive adipocyte degeneration occurs, often associated with insulin resistance and diabetes. Familial PLD has been mapped to 1q21-22 and in these studies, LMNA, encoding nuclear lamins A and C, was investigated as a positional candidate. Cao and Hegele looked at the coding region of LMNA in five probands from five Canadian kindreds segregating PLD. They found them all to be heterozygous for a G→A change at codon 482 in exon 8, resulting n the replacement of arginine (R) by guanine (Q). 1000 normal subjects showed no evidence for this change. They also sequenced the coding and flanking region of 11 other genes in the region but found no potential causative variants. Shackleton et al. identified five different missense mutations in LMNA among 10 families and three individuals with PLD. These included the R482Q mutation seen in the Canadian kindreds; two other changes at this residue (R482W and R482L); and 2 different mutations resulting in lysine to asparagine at the nearby residue (K486N). The affected residues are in the carboxy-terminal tail of the protein .

Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome. Bell DW, Varley JM, Szydlo TE, Kang DH, Wahrer DCR, Shannon KE, Lubratovich M, Verselis SJ, Isselbacher KJ, Fraumeni JF et al.: Science 1999, 286:2528-2531. • Significance: This paper details the discovery of a genetic defect that explains the incidence of Li-Fraumeni syndrome (LFS, a hereditary cancer susceptibilty syndrome) in patients with intact p53. The finding adds to our understanding of the molecular mechanism of tumorigenesis, and implicates hCHK2

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Sun C, Skaletsky H, Birren B, Devon K, Tang Z, Silber S, Oates R, Page DC: Nat Genet 1999 23:429-432. • Significance: This is the first identification of a gene on the Y chromosome that, when mutated, results in failure to make sperm. Findings: Infertility, caused by faulty sperm production, can be caused by deletion any one of three regions of the Y-chromosome – AZFa, AZFb or AZFc. Sun et al. sequenced the 0.8 Mb

gda2pap.qxd

03/22/2000

134

02:34

Page 134

Paper alert

AZFa region and identified two previously described functional genes (USP9Y and DBY) and 11 pseudogenes. They looked for mutations in USP9Y and DBY in the DNA of 576 infertile and 96 fertile men. five sequence variants were found, all in USP9Y. Four had no functional consequence and were also present in the father of the affected individual. One mutation, a 4 bp deletion in a splice-donor site, was de novo, and caused USP9Y protein truncation. A biopsy of the testis of the carrier of this mutation suggested a histological diagnosis of hyperspermatogenesis and spermatogenic arrest. These findings raise the possibility that mutations in USP9Y that lead to only a partial loss of function may underlie milder fertility problems in men Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis. Toomes C, Jame J, Wood AJ, Wu CL, McCormick D, Lench N, Hewitt C, Moynihan L, Roberts E, Woods G et al.: Nat Genet 1999, 23:421-424. • Significance: Finding the gene mutated in a severe, inherited form of gum disease (periodontitis) has implications for the cause, prevention and treatment of more common forms of periodontitis, which affects more than 30% of the population. Findings: Papillon-Lefevre syndrome (PLS) is a hereditary disorder characterised by severe early-onset periodontitis. The disease has previously been mapped to a 4–5 cM region on chromosome 11q14-q21. The authors used eight consanguineous families to refine the locus to a 1–2 cM region. The gene encoding the papain-like, lysosomal cysteine protease cathespin C (CTSC) was identified as a positional candidate within this region. They analysed the DNA of the affected members of the families and identified eight loss-of-function mutations in the gene, which were not present in control individuals. Assays of cathepsin C activity in family members with PLS revealed near-complete loss in affected individuals, and partial loss of activity in heterozygous carriers. It is suggested that the common forms of periodontitis might be caused by slight loss of cathepsin C activity.

Pattern formation and developmental mechanisms Selected by Mike Jones Chester Beatty Laboratories, London, UK

Turning of nerve growth cones induced by localized increases in intracellular calcium ions. Zheng JQ: Nature 2000, 403:89-93. AND

Calcium signalling in the guidance of nerve growth by netrin-1. Hong K, Nishiyama M, Henley J, Tessier-Lavigne M, Poo M: Nature 2000, 403:93-98. •• Significance: These papers provide direct evidence that localised calcium signalling directs growth cone turning and demonstrate that intracellular and extracellular calcium stores are necessary to mediate both attraction and repulsion to a gradient of the guidance molecule netrin-1. Findings: Localised increases in intracellular calcium, produced by focal laser-induced photolysis (FLIP) of caged calcium or by a microgradient of netrin-1, resulted in growth cones turning toward the side of higher calcium concentration. By lowering either the intracellular or extracellular calcium concentration, a similar localised increase in calcium concentration caused a repulsion of the growth cone. It is suggested that a focal calcium-mediated signal provides either attractive or

repulsive cues depending on the environment a growth cone senses. The organizer factors Chordin and Noggin are required for mouse forebrain development. Bachiller D, Klingensmith J, Kemp C, Belo JA, Anderson RM, May SR, McMahon JA, McMahon AP, Harland RM, Rossant J, De Robertis EM: Nature 2000, 403:658-661. • Significance: Evidence is presented which supports the idea that signalling by tissues derived from the mouse node, and not by the anterior visceral endoderm (AVE), are necessary for anterior patterning in the early embryo. Findings: Chordin and noggin are expressed in the node and axial mesendoderm. Double null mutant embryos develop with anterior truncations. Early markers of the AVE — including Hesx1, Lim1 and Cer-1 — are expressed normally, suggesting that the AVE is unaffected in the double mutants. Shh and HNF3β expression in the rostral midline is not detected in early somite stage mutants, suggesting that anterior mesendoderm is absent. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signalling cells. Burke R, Nellen D, Bellotto M, Hafen E, Senti K, Dickson BJ, Basler K: Cell 2000, 99:803-815. •• Significance: The authors describe the isolation of a novel gene, Dispatched, which functions to release cholesterol-modified Hedgehog (Hh) from Hedgehog-producing cells. (See also alert by T-sang et al., p0.) Findings: Dispatched encodes a protein (Disp) containing a sterol-sensing domain (similar to the Hh receptor Patched) and multiple putative transmembrane domains. Disp is required in the cells that produce cholesterol-modified Hh to promote release of the protein so that it may signal to neighbouring cells. It is suggested that Hh sequestration, mediated by Patched, and Hh release, mediated by Disp, both depend upon the cholesterol moiety attached to active Hh protein. Heritable and inducible genetic interference by doublestranded RNA encoded by transgenes. Tavernarakis N, Wang SL, Dorovkov M, Ryazanov A, Driscoll M: Nat Genet 2000, 24:180-183. • Significance: A transgenic method for producing doublestranded RNA which interferes with gene function (RNAi) in Caenorhabditis elegans that may be amenable to other model systems is described. Findings: Transgenes driving inverted repeat sequences are shown to be effective at disrupting gene function specifically. This system circumvents the problems associated with standard RNAi techniques and has advantages including the production of stable lines harbouring the blocking allele, the production of large numbers of mutants for biochemical analysis that can be made inducible to study the consequences of stage-specific gene inactivation. Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Tanimoto H, Itoh S, ten Dijke P, Tabata T: Mol Cell 2000, 5:59-71. • Significance: Suggests a novel patterning mechanism whereby the activity one of morphogen assists in shaping the functional domain of another. Findings: Phosphorylated Mothers against Decapentaplegic (DPP) (p-MAD) levels are highest near the source of DPP but

gda2pap.qxd

03/22/2000

02:34

Page 135

Paper alert

are low in anterior compartment cells of the wing disc which produce DPP. These decreased levels of p-MAD result from the actions of Hedgehog (Hh), which suppress the expression levels of thick veins (tkv), the receptor required for DPP signal transduction and which plays a role in limiting the range of DPP action. Hh therefore plays an indirect role in shaping the DPP gradient.

Differentiation and gene regulation Selected by Alison Schuldt Wellcome/CRC Institute, Cambridge, UK

Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Li L, Vaessin H: Genes Dev 2000, 14:147-151. •• Significance: In the central nervous system of Drosophila, one of the earliest signs of differentiation in neural precursors is the nuclear localisation of the homeodomain-containing transcription factor, Prospero. This paper demonstrates the link between nuclear localisation of Prospero and the decision to exit the mitotic state and undergo differentiation. Findings: Loss of Prospero results in increased mitotic activity. One consequence of this is prolonged expression of cell-cycle regulatory genes. Extra division is, in part, compensated for by increased cell death. Gal-4 -mediated expression of Prospero decreases mitotic activity and represses expression of cellcycle regulatory genes. The transcription factor Snail controls epithelial–mesencyhmal transitions by repressing E-cadherin expression. Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA: Nat Cell Biol 2000, 2:76-83. AND

The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Batle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, de Herreros AG: Nat Cell Biol 2000, 2:84-89. •• Significance: E-cadherin mediates cell–cell adhesion of epithelial cells. Loss of E-cadherin expression is associated with epidermal–mesenchmyal transitions during normal gastrulation and also when epithelial tumour cells become invasive. These two papers show that Snail, a transcription factor, represses Ecadherin expression during these transitions. This highlights Snail as a potential target for preventing tumour invasion. Findings: Both groups show that transfection of Snail represses expression of E-cadherin in mammalian epidermal cells. Batle et al. report that Snail binds E-cadherin directly through its E-box motifs. Cano et al. demonstrate that the ectopic expression of Snail in epithelial cells results in the acquisition of invasive properties. They further show that endogenous Snail is expressed in invasive regions of tumours. Sloppy paired acts as the downstream target of Wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits sloppy paired during neurogenesis. Bhat KM, Beers EHV, Bhat P: Development 2000, 127:655665. • Significance: Wingless (Wg) signalling acts to determine the fate of a particular neural precursor denoted NB4-2. Gsb-d (Gooseberry-distal), a homeodomain-containing transcription factor, antagonises this fate in the adjacent cell. This paper shows that sloppy-paired (slp), a transcription factor of the forkhead group, is a downstream target of wingless signalling, and

135

furthermore that Gsb-d blocks wingless signalling through antagonism of slp. Findings: NB4-2 is lost in slp mutants. Ectopic expression experiments suggest that slp acts downstream of wg. In the adjacent cell, overexpression of slp is sufficient to overcome repression by Gsb-d, allowing the cell to adopt a 4–2 fate. Antagonism of Notch signalling activity by members of a novel protein family encoded by the Bearded and Enhancer of split gene complex. Lai EC, Bodner R, Kavaler J, Freschi G, Posakony JW: Development 2000, 127:291-306. • Significance: Notch signalling mediates numerous cell-fate decisions during Drosophila development. This results in activation of the Enhancer of split complex through the transcription factor, Suppressor of Hairless (Su[H]). This paper identifies new targets for Su(H) and defines a novel gene family — the Bearded complex. Findings: The authors report seven new genes, all of which encode proteins containing small basic amphipathic α-helical domains, related to the Bearded gene. Each gene contains at least one binding site for Su(H). They find that the Bearded complex is expressed in regions of active Notch signalling. Overexpression of each gene modulates Notch signalling. Relief of gene repression by Torso RTK signalling: role of capicua in Drosophila terminal and dorsoventral patterning. Jimenez G, Guichet A, Ephrussi A, Casanova J: Genes Dev 2000, 14:224-231. • Significance: Torso (Receptor Tyrosine Kinase) signalling mediates differentiation of the terminal (head and tail) regions of the Drosophila embryo by inactivating a repressor of the terminal genes. Whereas Groucho (a DNA- binding protein) has been proposed to act as a co-repressor, the identity of the repressor itself was previously unknown. This paper identifies a new gene, capicua, a HMG box (high mobility group-box) containing transcription factor as this repressor. Findings: In cic mutants, terminal gene expression is expanded. Capicua binds to the co-repressor Groucho in vitro. In torso mutants, Cic protein is expressed in the terminal regions, showing that Cic is repressed post-transcriptionally by Torso signalling. Selected by Michael Tsang, Mizuki Azuma and Neil Hukriede National Institute of Child Health and Human Development, Bethesda, Maryland, USA

OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signalling pathways. Hata A, Seoane J, Lagna G, Montalvo E, Hemmati-Brivanlou A, Massagué J: Cell 2000, 100:229-240. •• Significance: This study describes the isolation of OAZ, a mutli-zinc finger transcription factor that associates and cooperates with Smad1/Smad4 activation of the Xvent2 reporter. Findings: In Xenopus, BMP-Smad signalling activates the expression of Xvent2. The authors identified the BMP2 responsive element (BRE) within the Xvent2 promoter. A yeast one-hybrid screen with a reporter containing multimers of the BRE element resulted in the isolation XOAZ, a 30 zinc finger transcription factor. XOAZ activated Xvent2 reporters only in the presence of BMP2 and synergistically activated Xvent2 reporters when Smad1 and Smad4 were co-transfected. Distinct zinc finger clusters were shown to bind to the Smad1/4 complex or to the BRE element, independently. In addition, previous studies revealed that rat OAZ can negatively regulate

gda2pap.qxd

03/22/2000

136

02:34

Page 136

Paper alert

differentiation of neuronal precursors in the olfactory epithelium by inhibiting the Olf signalling pathway through interactions with the Olf-1/EBF complex. In this study, the authors noted that the OLF-1/EBF zinc finger interaction cluster is dispensable for OAZ/Smad interactions. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signaling cells. Burke R, Nellen D, Bellotto M, Hafen E, Senti K-A, Dickson BJ, Basler K: Cell 1999, 99:803-815. •• Significance: During Drosophila limb development, cholesterol modified Hedgehog (Hh) is secreted from posterior compartment cells, and received in anterior compartment cells. Dispatched (Disp), a novel sterol sensing molecule potentiates the Hh signal through mediating the range of the secreted Hh molecule. (See also alert by Jones, p0.) Findings: Using a genetic screen to identify novel components of the Hh pathway, the authors identified a novel twelve pass transmembrane protein, disp. In the absence of disp, cholesterol-modified Hh is retained in posterior compartment cells, while cleaved, but non-cholesterol modified Hh can still diffuse to the anterior compartment cells. This suggests that Disp’s function is to release active Hh from the ‘sending’ cells. In addition, the Patched receptor and Disp share structural homology in the form of sterol-sensing domain (SSD), suggesting the common principle behind sending and receiving the Hh signal is through cholesterol modification. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Heanue TA, Reshef R, Davis RJ, Mardon G, Oliver G, Tomarev S, Lassar AB, Tabin CJ: Genes Dev 1999, 13:3231-3243. • Significance: It is known that eyeless, sine oculis, eyes absent, and dachshund (dac) form a gene network for Drosophila eye development. In the present study, the authors find that vertebrate homologs regulate chick myogenesis, suggesting conservation of these genes during organogenesis. Findings: The authors cloned chick Dachsund (Dach2) and demonstrated that Dach2 is capable of compensating for dac mutants during Drosophila eye formation. Expression of Dach2 overlaps in the limb muscle precursors with Pax3, Six1, and Eya2. Dach2 and Pax3 were shown to regulate each other expression using an in vitro somite culture system. Furthermore, the Dach2 and Eya2, or Eya2 and Six2 proteins physically interact to regulate Pax3 and other myogenic markers. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Fode C, Ma Q, Casarosa S, Ang S-L, Anderson DJ, Guillemot F: Genes Dev 2000, 14:67-80. • Significance: Mouse Neurogenin1(Ngn1), Neurogenin2 (Ngn2) and mouse achaete-scute (Mash1) are bHLH transcription factors involved in neuronal determination. In the present study, the authors demonstrate that the generation of specific neuronal types is dependent upon precise positioning of Ngn1, Ngn2 and Mash1. Findings: The relationship of Ngns expression in the dorsal domains of the telencephalon, with respect to Mash1 expression in the ventral region was investigated by using the Ngns and Mash1 mutants embryos. The Ngn2 and the double Ngns mutant embryos exhibited an up-regulation of Mash1 expression in dorsal telencephalon. This also resulted in a reduction in

expression of several dorsal neuronal markers, Tbr1 and Math1 and ectopic expression of ventral markers, Dlx1 and GAD67 within the dorsal domain. Moreover, forced over-expression of Mash1 in Ngn2-positive progenitors by a gene knock-in of Mash1 into the Ngn2 locus induced ventralization of dorsal telencephalon. These data suggest that Mash1 and the Ngns specify dorsal-ventral neuronal identity by regulating neurogenesis and regional patterning in the telencephalon. A novel cis-regulatory element, the PTS, mediates an antiinsulator activity in the Drosophila embryo. Zhou J, Levine M: Cell 1999, 99:567-575. •• Significance: Previously, it was unknown as to how certain enhancers could overcome strong insulators in directing gene expression. This study identifies a novel 625 bp ciselement, the promoter-targeting sequence (PTS), which can overcome enhancer blocking activities of two insulators, Fab-8 and su(Hw) and allow distal enhancers to activate gene transcription. Findings: During characterisation of an insulator region, the transvection mediating region (tmr) from the Abd-B locus, the authors uncovered an unexpected result. A minimal Fab-8 insulator blocked an enhancer from distal activating reporters, but by swapping the Fab8 insulator with the tmr insulator, one of the distal reporters was activated. Further analysis of the tmr insulator revealed a minimal 625 bp PTS that conferred the anti-insulator activity. PTS mutant embryos exhibit transformation of abdominal tergites, confirming that the PTS domain is important for regulating Abd-B gene expression.

Genomes and evolution Selected by Susan Douglas Institute for Marine Bioscience, Halifax, Nova Scotia, Canada

Carbonic anhydrase is an ancient enzyme widespread in prokaryotes. Smith KS, Jakubzick C, Whittam TS, Ferry JG: Proc Natl Acad Sci USA 1999, 96:15184-15189. • Significance: Carbonic anhydrase is ubiquitous in highly evolved eukaryotes but its occurrence in the Archaea and Bacteria is unknown. Genome sequencing of the methanoarchaea Methanobacterium thermoautotrophicum and Methanosarcina thermophila has led to the unexpected identification of a β-type carbonic anhydrase in the former and a new class of carbonic anhydrase (γ) in the latter. This has allowed the distribution of carbonic anhydrases among Bacteria and Archaea to be determined. Phylogenetic analyses of carbonic anhydrase sequences shows this to be an ancient enzyme that existed before the divergence of Archaea and Bacteria and played an important role in carbon dioxide fixation in early chemolithoautotrophic species. Findings: Cell extracts from metabolically diverse species from both the Archaea and Bacteria were assayed for carbonic anhydrase activity and cross-reacting proteins were identified using antibodies against the three classes of enzyme (α, β and γ). DNA and protein databases were searched for sequences with significant similarity to β-carbonic anhydrase from M. thermoautotrophicum or to the γ-carbonic anhydrase from M. thermophila and numerous matches were found among the Bacteria and Archaea. 26 matches were found for the β-type and 25 for the γ-type. In contrast, only eight matches were found to α-carbonic anhydrase in the Bacteria and none in the Archaea. Both β- and γ-type carbonic anhydrases were present in thermophiles representing the earliest branches in the universal tree of life. Divergence times were estimated and the root

gda2pap.qxd

03/22/2000

02:34

Page 137

Paper alert

of the γ-class was placed at 4.2 billion years ago, before the divergence of the Bacteria and Archaea. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima. Worning P, Jensen LJ, Nelson KE, Brunak S, Ussery DW: Nucleic Acids Res 2000, 28:706-709. • Significance: Periodicity analysis of DNA sequences from Bacteria, Archaea and Eukarya shows different periodicity values for the different domains and allows an estimation of lateral gene transfer that is independent of gene-sequence divergence. As much as a quarter of the genome of the bacterium Thermotoga maritima is proposed to have arisen by lateral tranfer from an Archaeal donor similar to Pyrococcus horikoshii. The implications of this phenomenon on phylogenetic inference are substantial. Findings: Periodicity spectra of 24 complete genomes were assessed using propeller twist, stacking energy and protein induced deformability (structural parameters) plus AT content. Although about one third of the genomes exhibited more than one period, the average spectra of the Bacterial genomes had a main peak at 11.3 bp whereas that of the Archaeal genomes was ~10 bp. All but three of the Bacterial genomes had values greater than or equal to the highest value of the Archaeal genomes. Archaea-like DNA present in the genome of T. maritima was obtained by aligning encoded proteins against proteins of five Archaeal genomes. Similarly, Bacteria-like DNA was obtained by alignment against proteins from six Bacterial genomes. Periodicity spectra of the Archaea-like DNA present in the T. maritima genome revealed two distinct peaks that matched those of the Archaea, P. horikoshii and the spectrum of the Bacteria-like sequences was very similar to the overall spectrum of T. maritima. Differences in periodicity could arise from differences in supercoiling or chromatin structure among the different domains. Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana. Lin X, Kaul S, Rounsley S, Shea TP, Benito MI, Town CD, Fujii CY, Mason T, Bowman CL, Barnstead M et al.: The Institute for Genomic Research: Nature 1999, 402:761-768. •• Significance: This and the accompanying paper (below) represent the first reports of large tracts of DNA from the small (130–140 Mb) Arabidopsis thaliana genome. Chromosome 2 comprises 15% of the genome and is presented as two contigs of 3.6 and 16 Mb. Within the centromere is a huge insertion of 270 kb region that is 99% identical to DNA corresponding to 75% of the Arabidopsis mitochondrial genome and represents the largest reported organellar-nuclear lateral transfer. In addition, 135 putative choroplast-derived genes are scattered along the chromosome. Findings: Chromosomes 2 and 4 are both acrocentric with the short arms containing rDNA repeats arranged head-to-tail towards the centromere. Four large blocks of genes are duplicated between the two chromosomes. Chromosome 2 possesses 4,037 predicted genes of which 51.5% have been assigned functions. The average gene density is 4.4 kb/gene and the average exon content is 4.6/gene, resulting in intergenic sequences comprising >50% of the chromosome. Over half of the predicted proteins have a significant match to at least one other protein encoded on chromosome 2, and these are often found within tandem duplications or in large chromosomal duplications. Most of the duplications have occurred after the separation of plants from the animal and fungal lineages; >65% of the predicted genes do not show significant similarity to any other completed genome and are probably unique to plants.

137

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana. The European Union Arabidopsis Genome Sequencing Consortium & The Cold Spring Harbor, Washington University in St. Louis and PE Biosystems Arabidopsis Sequencing Consortium: Nature 1999, 402:769-777. •• Significance: Chromosome 4 represents 17% of the genome and is presented as two contigs of 2.6 and 14.5 Mb and a smaller centromeric contig of 0.28 Mb. Sequence analysis reveals numerous genes with homologs in human and Caenorhabditis elegans genomes, many of which are involved with processes characteristic of multicellular eukaryotes such as cellular communication, defence and disease. This work provides a solid foundation for further studies designed to define the functions of these genes and augment our understanding of basic plant processes. Findings: Chromosome 4 possesses 3744 predicted genes of which 60% have been assigned functions. Although 34% of the predicted genes match expressed sequence tags, only 6% of the genes are highly expressed in the tissues sampled. Approximately 18% of predicted proteins contain amino-terminal extensions typical of mitochondrial and chloroplast transit peptides, and represent lateral gene transfers from organellar genomes. 12% of genes with predicted functions are present as clusters, most of which are in adjacent regions of the chromosome. The gene density is 4.6 kb/gene except in the pericentromeric heterochromatin which is characterised by very low gene density and recombination rate, and high frequency of retroelements and transposons, many of which are degenerate. Probable pseudogenes and genes with low expression levels are also typical of this region. Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capuslatus. Lang AS, Beatty JT: Proc Natl Acad Sci USA 2000, 97:859-864. • Significance: This paper describes the sequence and functional analysis of a 15 kb gene cluster encoding a genetic transfer agent (GTA) from Rhodobacter capsulatus. This genetic element, embedded in the R. capuslatus genome, is responsible for the transfer of random 4.5 kb segments from the host genome into recipient cells and may be an ancient prophage remnant that has co-evolved with its host to enhance genetic exchange under adverse environmental conditions. Findings: Using Tn5 insertion mutagenesis, a 30 kb region of the R. capuslatus genome was identified that participated in the production of GTA particles. Sequence analysis of this region revealed a cluster of open reading frames with significant amino acid similarity to known prophage proteins. Upstream of this cluster are genes for a transcriptional regulator (ctrA), DNA ligase (dnaJ) and DNA recombination (recG). Transcription of GTA genes is increased during stationary phase resulting in production of particles that transduce genomic DNA fragments thereby contributing genetic diversity to the cell population. This genetic exchange apparently has been conserved during evolution as strains of R. capsulatus from geographically disperse locations produce GTA. Insertion of a transcriptionally congruent KIXX cartridge into ctrA and insertion of TN5 into cckA (a gene required for the activation of CtrA and located elsewhere in the host genome) impaired GTA production, indicating that these host genes are involved in positive regulation of the genetic transfer process and constitute a sensor kinase/response regulator system sensitive to growth-limiting conditions.