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The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to rats signaling
WONITOR
The human PAX6gene is mutated in two patients with aniridia T. JORDAN E T A L
N a t u r e Genet. 1,328-332
Early this year, the human genetic disease Waardenburg syndrome type I (WSI) was shown to be caused by mutations in the human homologue of the mouse Pax-3 gene, confirming the suspicion that WSI might be similar to the mouse Splotch mutant, which results from P a x - 3 mutations. It was tempting at the time to suggest that there might be a similar link-up between the mouse small-eye mutation, associated with mutations in Pax-6,
Analysing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models E.M. SOUTHERN E T A L
G e n o m i c s 13, 1008-1017
Dideoxy chain-termination sequencing is currently the method of choice even for large-scale sequencing projects. But there is certainly a place for alternatives based on technologies that are sufficiently different from chain termination to have complementary strengths and weaknesses. Southern et aL describe a method for synthesizing complete sets of oligonucleotides of defined length in systematic arrays, covalently attached to a glass plate. The approach used is
The SH2 and SH3 domain-containing protein GRB2links receptor tyrosine kinases to ras signaling E.J. LOWENSTEINE T A L
Cell 70, 431-442
Experiments with antibody microinjection and dominant negative mutants show that tyrosine kinase growth factor receptors require cellular p21 ras function to stimulate DNA synthesis. Thus there has to be a link between tyrosine kinases and Ras. Schlessinger's laboratory have now obtained suggestive evidence that a protein containing little more than two Src homology 3 (SH3) domains and one SH2 domain is such a link. By screening expression -libraries with the phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor they have identified a
and the human condition aniridia. Jordan et al. now show that this is indeed the case. A candidate gene for aniridia, isolated by positional cloning from a region of chromosome 11p13 near the Wilms' Tumour predisposition gene WT1, proved to be the human P A X 6 gene. Using a PCRbased strategy, Jordan et al. found mutations in the P A X 6 gene in two aniridia cell lines. One, a two-base insertion in the gene, was predicted to lead to production of a truncated protein. The second, which was detected as an RNA lacking an exon, appeared to be a splicing mutation resulting from an as yet undetected
point mutation or very small deletion. The product of the mutant RNA would lack part of the PAX6 homeodomain. Although the association between P A X 6 mutations and aniridia now seems incontrovertible, there is evidence that mutations at a second human locus, AN2, may be responsible for some cases of aniridia. It remains to be determined whether AN2 is a second aniridia gene, or has a more indirect effect on development of the condition. Genetic modifying factors do appear to affect the small-eye phenotype in the mouse, so perhaps the mouse model will be a helpful starting point here too. ~z
formally similar to the familiar way of writing the 64 codon triplets in rows and columns. A plate featuring all octapurines was used to explore the possibility of sequencing by annealing sequences of interest to systematic arrays and assembling the sequence from the positives. (An array of all 8-mers should theoretically enable the unambiguous identification of a sequence of up to 200 bases.) Data analysis and sequence assembly are complex but computer based: the hybridization pattern is detected by phosphor imaging, and the signal is normalized by comparison with the same grid hybridized with degenerate oligonucleotides. This minimizes the effects of variable oligonucleotide
yield, and the different base composition and sequence of the oligonucleotides. Sequence assembly involves comparing the observed pattern with the patterns simulated for all possible sequences of the length of the subject sequence. 'The sequence' is that which best fits the observed data. Arguably this is a strength of the approach; every sequence comes with a statistical measure of the quality of the assignment. But the method, which should be easy to automate, will undoubtedly be useful for sequence comparison and mutation detection: anneal sequences with a single base change to 8-mers and then there will be eight differences in the hybridization pattern.
number of SH2-containing proteins. One of these, GRB2, is highly homologous to the protein encoded by the C. elegans sere-5 gene. Genetic studies argue that the s e m - 5 gene product is involved in the signalling between a tyrosine kinase receptor - Let-23 and Ras. Lowenstein et al. show that GRB2 binds to tyrosine-phosphorylated EGF and PDGF receptors via its SH2 domain. Strikingly, microinjection of GRB2 together with normal nononcogenic p21 ras into quiescent fibroblasts enhances DNA synthesis much more than injection of Ras protein alone. Since normal p21 ras has to be converted to the active GTP-bound form to induce DNA synthesis, this result suggests that increasing the level of GRB2 promotes the conversion of p21 ras to the GTP-bound form. A likely scenario for such an
effect is that GRB2 somehow binds to an exchange factor for Ras in the cytosol, and after autophosphorylation of the receptor tyrosine kinase, the complex of GRB2 and exchange factor is brought to the membrane by the interaction between the SH2 domain of GRB2 and the tyrosinephosphorylated receptor. Juxtaposition of p21 ras and the exchange factor at the membrane then permits GTP exchange to take place. Intriguingly, the SH3 domains are required for the activity of GRB2, since point mutations in either SH3 domain block the effect of GRB2. With the recent cloning of a number of guanine nucleotide exchange factors for Ras, these experiments point the way to understanding how tyrosine kinase activity promotes the activation of p21 ras to the GTP-bound state. /z
TIG NOVEMBER 1 9 9 2 VOL. 8 NO. 11
The human PAX6gene is mutated in two patients with aniridia T. JORDAN E T A L
N a t u r e Genet. 1,328-332
Early this year, the human genetic disease Waardenburg syndrome type I (WSI) was shown to be caused by mutations in the human homologue of the mouse Pax-3 gene, confirming the suspicion that WSI might be similar to the mouse Splotch mutant, which results from P a x - 3 mutations. It was tempting at the time to suggest that there might be a similar link-up between the mouse small-eye mutation, associated with mutations in Pax-6,
Analysing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models E.M. SOUTHERN E T A L
G e n o m i c s 13, 1008-1017
Dideoxy chain-termination sequencing is currently the method of choice even for large-scale sequencing projects. But there is certainly a place for alternatives based on technologies that are sufficiently different from chain termination to have complementary strengths and weaknesses. Southern et aL describe a method for synthesizing complete sets of oligonucleotides of defined length in systematic arrays, covalently attached to a glass plate. The approach used is
The SH2 and SH3 domain-containing protein GRB2links receptor tyrosine kinases to ras signaling E.J. LOWENSTEINE T A L
Cell 70, 431-442
Experiments with antibody microinjection and dominant negative mutants show that tyrosine kinase growth factor receptors require cellular p21 ras function to stimulate DNA synthesis. Thus there has to be a link between tyrosine kinases and Ras. Schlessinger's laboratory have now obtained suggestive evidence that a protein containing little more than two Src homology 3 (SH3) domains and one SH2 domain is such a link. By screening expression -libraries with the phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor they have identified a
and the human condition aniridia. Jordan et al. now show that this is indeed the case. A candidate gene for aniridia, isolated by positional cloning from a region of chromosome 11p13 near the Wilms' Tumour predisposition gene WT1, proved to be the human P A X 6 gene. Using a PCRbased strategy, Jordan et al. found mutations in the P A X 6 gene in two aniridia cell lines. One, a two-base insertion in the gene, was predicted to lead to production of a truncated protein. The second, which was detected as an RNA lacking an exon, appeared to be a splicing mutation resulting from an as yet undetected
point mutation or very small deletion. The product of the mutant RNA would lack part of the PAX6 homeodomain. Although the association between P A X 6 mutations and aniridia now seems incontrovertible, there is evidence that mutations at a second human locus, AN2, may be responsible for some cases of aniridia. It remains to be determined whether AN2 is a second aniridia gene, or has a more indirect effect on development of the condition. Genetic modifying factors do appear to affect the small-eye phenotype in the mouse, so perhaps the mouse model will be a helpful starting point here too. ~z
formally similar to the familiar way of writing the 64 codon triplets in rows and columns. A plate featuring all octapurines was used to explore the possibility of sequencing by annealing sequences of interest to systematic arrays and assembling the sequence from the positives. (An array of all 8-mers should theoretically enable the unambiguous identification of a sequence of up to 200 bases.) Data analysis and sequence assembly are complex but computer based: the hybridization pattern is detected by phosphor imaging, and the signal is normalized by comparison with the same grid hybridized with degenerate oligonucleotides. This minimizes the effects of variable oligonucleotide
yield, and the different base composition and sequence of the oligonucleotides. Sequence assembly involves comparing the observed pattern with the patterns simulated for all possible sequences of the length of the subject sequence. 'The sequence' is that which best fits the observed data. Arguably this is a strength of the approach; every sequence comes with a statistical measure of the quality of the assignment. But the method, which should be easy to automate, will undoubtedly be useful for sequence comparison and mutation detection: anneal sequences with a single base change to 8-mers and then there will be eight differences in the hybridization pattern.
number of SH2-containing proteins. One of these, GRB2, is highly homologous to the protein encoded by the C. elegans sere-5 gene. Genetic studies argue that the s e m - 5 gene product is involved in the signalling between a tyrosine kinase receptor - Let-23 and Ras. Lowenstein et al. show that GRB2 binds to tyrosine-phosphorylated EGF and PDGF receptors via its SH2 domain. Strikingly, microinjection of GRB2 together with normal nononcogenic p21 ras into quiescent fibroblasts enhances DNA synthesis much more than injection of Ras protein alone. Since normal p21 ras has to be converted to the active GTP-bound form to induce DNA synthesis, this result suggests that increasing the level of GRB2 promotes the conversion of p21 ras to the GTP-bound form. A likely scenario for such an
effect is that GRB2 somehow binds to an exchange factor for Ras in the cytosol, and after autophosphorylation of the receptor tyrosine kinase, the complex of GRB2 and exchange factor is brought to the membrane by the interaction between the SH2 domain of GRB2 and the tyrosinephosphorylated receptor. Juxtaposition of p21 ras and the exchange factor at the membrane then permits GTP exchange to take place. Intriguingly, the SH3 domains are required for the activity of GRB2, since point mutations in either SH3 domain block the effect of GRB2. With the recent cloning of a number of guanine nucleotide exchange factors for Ras, these experiments point the way to understanding how tyrosine kinase activity promotes the activation of p21 ras to the GTP-bound state. /z
TIG NOVEMBER 1 9 9 2 VOL. 8 NO. 11