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Mutant lac repressors with new specificities hint at rules for protein-DNA interactions
~ONITOR Mutantlac repressors with new speclficities hint at rules for protein-DNAinteractions N. LEHMING ETAL.
EMBOJ. 9, 615-621
The three operators of the tac operon cooperate in repression S. OEHLER, E.R. EISMANN, H. KR~ER AND B. MOLLER-HILL
EMBOJ. 9, 973--979 Our view of transcriptional control by protein-DNA binding has been shaped largely by work on the E. coli la¢ operon, and two recent papers from MOiler-Hill and colleagues indicate that it still has things to teach us. The tetrameric/ac repressor interacts with the major groove of DNA through an a-helix. Lehmir~g et al. have developed a two-plasmid system that allows them to vary (1) the
Genetic analysis of defecation in Caelorhabditis ele&atts J.H. THOMAS
Genetics 124, 855--872 The nervous system of Caenorbabditis eleg,ans can, to a remarkable extent, be dispensed with under laboratory conditions. Thus classic mutation analysis can be used to identify genes important in neural function in particular aspects of nematode behaviour (e.g. locomotion, touch sensitivity, etc.). Thomas has now extended this repertoire to defecation. Defecation in C. elegans relies on three sets of muscles, which act cyclically to expel intestinal contents: first, a set of posterior body muscles (pBoc) contract, then a set of anterior body muscles (aBoc) contract, and finally Exp muscles open the anus and force out the faeces. The 18 genes identified fall into five groups: mutations in two specifically affect pBoc, four the aBoc, four the Exp muscles and six both aBoc and Exp; mutations in the other two affect the timing of defecation but not its mechanics. The three sets of muscles probably act independendy, since abolition of one muscle function may not affect the other steps. Furthermore, most of the mutations probably affect control of the muscle tissue, since contraction is still observed in other behaviours (e.g. locomotion) that the muscle is involved in. Thomas has constructed a genetic pathway on the basis of these mutations and their interactions, and suggests that the five groups of mutations may affect different classes of neurons; each class controls a particular aspect of muscle function. Laser ablation experiments on these hypothetical neurons would help to test this idea.
lac operator sequence and (2) the lac repressor carried by cells, in order to determine the strength of repression for different operatorrepressor combinations in vfvo. Residues 1 and 2 of the recognition helix are known to interact with base pairs 4 and 5 of the operator, and 86 possible amino acid/base pair combinations were tested. Generally, interfictions were additive, allowing the contribution of each amino acid/base pair interaction to be calculated independently, and the repressive powers of other combinations to be predicted. Interestingly, the gai promoter follows similar additive rules, which may be applicable to other prokaryotic proteins binding to DNA through an ct-helix. The lac operon is regulated extremely effectively: in the presence of inducer, /ac enzyme levels increase
1000-fold, compared with gal operon induction of about 20-fold. Oehler et al. analysed the effects of the three lac operators (01 and the low-affinity 'pseudo-operators' 02 and 03). Surprisingly, in the absence of both 02 and 03, repression drops by 50fold, indicating that these operators are highly influential in gene expression. Moreover, DNA looping is an essential part of their action, a point emphasized by the use of the mutant l a d gene iadt (i adi encodes a dimeric repressor that can bind a single operator, but cannot loop out DNA by binding a pair of operators). For 03 , looping may interfere with the binding of catabolite activator protein. Thus the /ac operon is clearly more complicated than we first imagined, and may give us more clues about how other repressors function.a~
The Xenopus MyoDgene. an unlocalized maternal mRNApredates lineage-restricted expression in the early embryo
genes. MyoD mRNA first appears during Xenopus oogenesis, and, curiously, it is not restricted to regions destined to become musde. Zygotic mRNA accumulates from the mid-blastula transition onwards, and is restricted to somitic mesodeml; expression can also be induced by treatment of blastula animal pole explants with mesoderminducing factors. Early expression of MyoD and its induction by mesoderm-inducing factors lends weight to the theory that it is involved in the first steps of muscle commitment. The widespread distribution of the maternal transcript is more mysterious, but may be responsible for the flexibility required to cope with the variability in the position of mesoderm induction, the exact position of which will depend on the site of sperm entry.
R.P. HARVEY
Development 108, 669-680 Four related genes - myd, Myf-5, myogenin and M y o D - effectively convert mouse 10T1/2 fibroblasts into myoblasts, and are thus considered to be instrumental in muscle cell differentiation. In Xenopus, muscle differentiation begins during mesoderm induction, and it would be interesting to know how the Xenopus homologues of these genes act during development. Harvey screened a Xenopus cDNA library for MyoD-like genes, and isolated two MyoD cDNAs that probably derive from duplicated
Selective enrichment of a large size genomic DNAfragmentby affinity capture, an approachfor genome mapping R.P. KANDPAL,D.C. WARDAND S.M. WEISSMAN
Nucleic Acids Res. 18, 1789-1795 YACs (yeast artificial chromosomes) permit the cloning of DNA fragments hundreds of kb in size, and promise to be invaluable in the construction of a large scale restriction map of the human genome. However, there is as yet no way to chose direcdy which DNA fragments to clone, and YACs may have other shortcomings (e.g. some sequences may be refractory to cloning). Kandpal et al. have developed an adjunct to YAC cloning chromosome 'fishing' - that allows enrichment of specific DNA fragments. DNA is digested with a rarecutting enzyme, and is then made single stranded at its ends by sparing TtG JUNE 1990 rot-. 6 NO. 6
at
use of an exonuclease. This is annealed with an RNA containing (1) sequences complementary to the end of the fragment and (2) a probe sequence; a second, biotinylated RNA is annealed to the probe sequences and the resulting tripartite complex is separated by passing it over an avidin-coated matrix. Ribonuclease is used to free the DNA fragment. Kandpal et al. isolated a 150 kb SfiI fragment from the ~-globin gene cluster by this method, consistently achieving 350-fold enrichment. Since this technique requires nucleotide sequences around the rare-cLotting site, a linking library (comprising DNA fragments containing rare-cutting sites) is a must, and such libraries are becoming more common. For genome mappil~.~, further purification could allow direct sequencing of the fragment, or isolation of cDNAs of genes encoded within it.
EMBOJ. 9, 615-621
The three operators of the tac operon cooperate in repression S. OEHLER, E.R. EISMANN, H. KR~ER AND B. MOLLER-HILL
EMBOJ. 9, 973--979 Our view of transcriptional control by protein-DNA binding has been shaped largely by work on the E. coli la¢ operon, and two recent papers from MOiler-Hill and colleagues indicate that it still has things to teach us. The tetrameric/ac repressor interacts with the major groove of DNA through an a-helix. Lehmir~g et al. have developed a two-plasmid system that allows them to vary (1) the
Genetic analysis of defecation in Caelorhabditis ele&atts J.H. THOMAS
Genetics 124, 855--872 The nervous system of Caenorbabditis eleg,ans can, to a remarkable extent, be dispensed with under laboratory conditions. Thus classic mutation analysis can be used to identify genes important in neural function in particular aspects of nematode behaviour (e.g. locomotion, touch sensitivity, etc.). Thomas has now extended this repertoire to defecation. Defecation in C. elegans relies on three sets of muscles, which act cyclically to expel intestinal contents: first, a set of posterior body muscles (pBoc) contract, then a set of anterior body muscles (aBoc) contract, and finally Exp muscles open the anus and force out the faeces. The 18 genes identified fall into five groups: mutations in two specifically affect pBoc, four the aBoc, four the Exp muscles and six both aBoc and Exp; mutations in the other two affect the timing of defecation but not its mechanics. The three sets of muscles probably act independendy, since abolition of one muscle function may not affect the other steps. Furthermore, most of the mutations probably affect control of the muscle tissue, since contraction is still observed in other behaviours (e.g. locomotion) that the muscle is involved in. Thomas has constructed a genetic pathway on the basis of these mutations and their interactions, and suggests that the five groups of mutations may affect different classes of neurons; each class controls a particular aspect of muscle function. Laser ablation experiments on these hypothetical neurons would help to test this idea.
lac operator sequence and (2) the lac repressor carried by cells, in order to determine the strength of repression for different operatorrepressor combinations in vfvo. Residues 1 and 2 of the recognition helix are known to interact with base pairs 4 and 5 of the operator, and 86 possible amino acid/base pair combinations were tested. Generally, interfictions were additive, allowing the contribution of each amino acid/base pair interaction to be calculated independently, and the repressive powers of other combinations to be predicted. Interestingly, the gai promoter follows similar additive rules, which may be applicable to other prokaryotic proteins binding to DNA through an ct-helix. The lac operon is regulated extremely effectively: in the presence of inducer, /ac enzyme levels increase
1000-fold, compared with gal operon induction of about 20-fold. Oehler et al. analysed the effects of the three lac operators (01 and the low-affinity 'pseudo-operators' 02 and 03). Surprisingly, in the absence of both 02 and 03, repression drops by 50fold, indicating that these operators are highly influential in gene expression. Moreover, DNA looping is an essential part of their action, a point emphasized by the use of the mutant l a d gene iadt (i adi encodes a dimeric repressor that can bind a single operator, but cannot loop out DNA by binding a pair of operators). For 03 , looping may interfere with the binding of catabolite activator protein. Thus the /ac operon is clearly more complicated than we first imagined, and may give us more clues about how other repressors function.a~
The Xenopus MyoDgene. an unlocalized maternal mRNApredates lineage-restricted expression in the early embryo
genes. MyoD mRNA first appears during Xenopus oogenesis, and, curiously, it is not restricted to regions destined to become musde. Zygotic mRNA accumulates from the mid-blastula transition onwards, and is restricted to somitic mesodeml; expression can also be induced by treatment of blastula animal pole explants with mesoderminducing factors. Early expression of MyoD and its induction by mesoderm-inducing factors lends weight to the theory that it is involved in the first steps of muscle commitment. The widespread distribution of the maternal transcript is more mysterious, but may be responsible for the flexibility required to cope with the variability in the position of mesoderm induction, the exact position of which will depend on the site of sperm entry.
R.P. HARVEY
Development 108, 669-680 Four related genes - myd, Myf-5, myogenin and M y o D - effectively convert mouse 10T1/2 fibroblasts into myoblasts, and are thus considered to be instrumental in muscle cell differentiation. In Xenopus, muscle differentiation begins during mesoderm induction, and it would be interesting to know how the Xenopus homologues of these genes act during development. Harvey screened a Xenopus cDNA library for MyoD-like genes, and isolated two MyoD cDNAs that probably derive from duplicated
Selective enrichment of a large size genomic DNAfragmentby affinity capture, an approachfor genome mapping R.P. KANDPAL,D.C. WARDAND S.M. WEISSMAN
Nucleic Acids Res. 18, 1789-1795 YACs (yeast artificial chromosomes) permit the cloning of DNA fragments hundreds of kb in size, and promise to be invaluable in the construction of a large scale restriction map of the human genome. However, there is as yet no way to chose direcdy which DNA fragments to clone, and YACs may have other shortcomings (e.g. some sequences may be refractory to cloning). Kandpal et al. have developed an adjunct to YAC cloning chromosome 'fishing' - that allows enrichment of specific DNA fragments. DNA is digested with a rarecutting enzyme, and is then made single stranded at its ends by sparing TtG JUNE 1990 rot-. 6 NO. 6
at
use of an exonuclease. This is annealed with an RNA containing (1) sequences complementary to the end of the fragment and (2) a probe sequence; a second, biotinylated RNA is annealed to the probe sequences and the resulting tripartite complex is separated by passing it over an avidin-coated matrix. Ribonuclease is used to free the DNA fragment. Kandpal et al. isolated a 150 kb SfiI fragment from the ~-globin gene cluster by this method, consistently achieving 350-fold enrichment. Since this technique requires nucleotide sequences around the rare-cLotting site, a linking library (comprising DNA fragments containing rare-cutting sites) is a must, and such libraries are becoming more common. For genome mappil~.~, further purification could allow direct sequencing of the fragment, or isolation of cDNAs of genes encoded within it.