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Objective To evaluate the clinical performance of non‐invasive prenatal testing for trisomy 21, 18, and 13 using targeted cell‐free DNA (cfDNA) analysis. Methods Targeted cfDNA analysis using DANSR™ and FORTE™ with microarray quantitation was used to evaluate the risk of trisomy 21, 18, and 13 in blinded samples from 799 singleton, twin, natural, and IVF pregnancies. Subjects either had fetal chromosome evaluation by karyotype, FISH, QF‐PCR, or karyotype for newborns with suspected aneuploidy at birth. The results of targeted cfDNA analysis were compared to clinical genetic testing outcomes to assess clinical performance. Results Targeted cfDNA analysis with microarray quantification identified 107/108 trisomy 21 cases (99.1%), 29/30 trisomy 18 cases (96.7%), and 12/12 trisomy 13 cases (100%). The specificity was 100% for all three trisomies. Combining this data with all published clinical performance studies using DANSR/FORTE methodology for greater than 23 000 pregnancies, the sensitivity of targeted cfDNA analysis was calculated to be greater than 99% for trisomy 21, 97% for trisomy 18, and 94% for trisomy 13. Specificity for each trisomy was greater than 99.9%. Conclusion Targeted cfDNA analysis demonstrates consistently high sensitivity and extremely low false positive rates for common autosomal trisomies in pregnancy across quantitation platforms. © 2015 Ariosa Diagnostics Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd., What's already known about this topic? NIPT using targeted cfDNA analysis with NGS has high sensitivity and specificity for fetal trisomy 21 and other autosomal trisomies. What does this study add? This study establishes the high sensitivity and specificity of NIPT using targeted cfDNA analysis with a microarray quantitation platform, and demonstrates that clinical performance is based on the targeted cfDNA analysis method rather than the quantitation method, as the performance using microarray is comparable with performance from previous NGS studies.

Objective: To determine the performance of a targeted microarray-based cell-free DNA (cfDNA) test (Harmony Prenatal Test®) for the identification of pregnancies at increased risk for 22q11.2 deletion. Methods: Test performance was determined in 2 steps including a total of 1,953 plasma samples. Analytical validation was performed in 1,736 plasma samples. Clinical verification of performance was performed in an additional 217 prospectively ascertained samples from pregnancies with fetal deletion status determined by diagnostic testing. Results: Analytical sensitivity was 75.4% (95% CI: 67.1-82.2%) based on 122 samples with deletions ranging from 1.96 to 3.25 Mb. In 1,614 presumed unaffected samples, specificity was determined to be at least 99.5% (95% CI: 99.0-99.7%). In the clinical cohort, 5 of 7 samples from pregnancies affected with 22q11.2 deletion were determined to have a high probability of deletion. There were no false positive results in the 210 unaffected samples in this cohort. These clinical data are consistent with the performance demonstrated in the analytical validation. Conclusions: cfDNA testing using a targeted microarray-based technology is able to identify pregnancies at increased risk for 22q11.2 deletions of 3.0 Mb and smaller while maintaining a low false positive rate., SCOPUS: ar.j, info:eu-repo/semantics/published

See related article by Sparks et al., [Cancer Res 1998;58:1130–4][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In the mid 1990s, the human genome project was well underway, but it would still be more than 5 years before the first draft of the human genome was published and more

Objective To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18). Methods Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR ™ ). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. Results At the lowest sequencing depth, corresponding to 204000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches. Conclusion Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.

Toward $1000 Genomes The ability to generate human genome sequence data that is complete, accurate, and inexpensive is a necessary prerequisite to perform genome-wide disease association studies. Drmanac et al. (p. 78 , published online 5 November) present a technique advancing toward this goal. The method uses Type IIS endonucleases to incorporate short oligonucleotides within a set of randomly sheared circularized DNA. DNA polymerase then generates concatenated copies of the circular oligonucleotides leading to formation of compact but very long oligonucleotides which are then sequenced by ligation. The relatively low cost of this technology, which shows a low error rate, advances sequencing closer to the goal of the $1000 genome.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene “mountains” and a much larger number of gene “hills” that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

A remaining challenge for the human genome project involves the identification and annotation of expressed genes. The public and private sequencing efforts have identified approximately 15,000 sequences that meet stringent criteria for genes, such as correspondence with known genes from humans or other species, and have made another approximately 10,000-20,000 gene predictions of lower confidence, supported by various types of in silico evidence, including homology studies, domain searches, and ab initio gene predictions. These computational methods have limitations, both because they are unable to identify a significant fraction of genes and exons and because they are unable to provide definitive evidence about whether a hypothetical gene is actually expressed. As the in silico approaches identified a smaller number of genes than anticipated, we wondered whether high-throughput experimental analyses could be used to provide evidence for the expression of hypothetical genes and to reveal previously undiscovered genes. We describe here the development of such a method--called long serial analysis of gene expression (LongSAGE), an adaption of the original SAGE approach--that can be used to rapidly identify novel genes and exons.

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.

DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.

Inactivation of the adenomatous polyposis coli ( APC ) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by β-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of β-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of β-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or β-catenin.

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.

Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

OBJECTIVE: We sought to evaluate performance of a noninvasive prenatal test for fetal trisomy 21 (T21) and trisomy 18 (T18). STUDY DESIGN: A multicenter cohort study was performed whereby cell-free DNA from maternal plasma was analyzed. Chromosomeselective sequencing on chromosomes 21 and 18 was performed with reporting of an aneuploidy risk (High Risk or Low Risk) for each subject. RESULTS: Ofthe81T21cases,allwereclassifiedasHighRiskforT21 andtherewas1false-positiveresultamongthe2888normalcases,for asensitivityof100%(95%confidenceinterval[CI],95.5‐100%)anda false-positive rate of 0.03% (95% CI, 0.002‐0.20%). Of the 38 T18 cases, 37 were classified as High Risk and there were 2 false-positive results among the 2888 normal cases, for a sensitivity of 97.4% (95% CI, 86.5‐99.9%) and a false-positive rate of 0.07% (95% CI, 0.02‐0.25%). CONCLUSION: Chromosome-selective sequencing of cell-free DNA and application of an individualized risk algorithm is effective in the detection of fetal T21 and T18.

We sought to develop a novel biochemical assay and algorithm for the prenatal evaluation of risk for fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA obtained from maternal blood.We assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies. We used digital analysis of selected regions in combination with a novel algorithm, fetal-fraction optimized risk of trisomy evaluation (FORTE), to determine trisomy risk for each subject.In all, 163/171 subjects in the training set passed quality control criteria. Using a Z statistic, 35/35 T21 cases and 7/7 T18 cases had Z statistic3 and 120/121 disomic cases had Z statistic3. FORTE produced an individualized trisomy risk score for each subject, and correctly discriminated all T21 and T18 cases from disomic cases. All 167 subjects in the blinded validation set passed quality control and FORTE performance matched that observed in the training set correctly discriminating 36/36 T21 cases and 8/8 T18 cases from 123/123 disomic cases.Digital analysis of selected regions and FORTE enable accurate, scalable noninvasive fetal aneuploidy detection.

To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.At the lowest sequencing depth, corresponding to 204,000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410,000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620,000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment.

The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

The adenomatous polyposis coli gene ( APC ) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of β-catenin, which then binds T cell factor–4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c- MYC oncogene is identified as a target gene in this signaling pathway. Expression of c- MYC was shown to be repressed by wild-type APC and activated by β-catenin, and these effects were mediated through Tcf-4 binding sites in the c- MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.

Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.

This chapter describes screening phage-displayed random peptide libraries. Screening of phage-displayed random peptide libraries represents a powerful means of identifying peptide ligands for targets of interest. Fundamentally, the screening of phage-displayed random peptide libraries for binding peptides includes the steps such as obtaining a suitable library and source of target, and incubating the library with target and capturing binding phage. It is found that in addition to being used as a vector for accessible expression of peptide libraries, M 13 has been used to display mutant libraries of entire proteins, including antibodies and growth hormone. To minimize problems arising from instability of phage-displayed peptides, phage should be protected from proteolytic degradation. Displayed peptides are susceptible to degradation during phage propagation in a bacterial culture. Elution of phage from the target:phage complex is an important aspect of screening phage-displayed random peptide libraries. One round of affinity purification typically affects the recovery of approximately 1% of PFU representing any given binding clone. The number of phage particles representing any given binding clone that are recovered from the first round is reduced to the point that binding clones may be lost if subjected to a second round of purification without intervening amplification. It is found that enrichment of binding phage may be assessed by doping a library aliquot with an equal number of nonbinding phage which is distinguishable from library phage.

The F-type subfamily of LINE-1 or L1 retroposons [for long interspersed (repetitive) element 1] was dispersed in the mouse genome several million years ago. This subfamily appears to be both transcriptionally and transpositionally inactive today and therefore may be considered evolutionarily extinct. We hypothesized that these F-type L1s are inactive because of the accumulation of mutations. To test this idea we used phylogenetic analysis to deduce the sequence of a transpositionally active ancestral F-type promoter, resurrected it by chemical synthesis, and showed that it has promoter activity. In contrast, F-type sequences isolated from the modern genome are inactive. This approach, in which the automated DNA synthesizer is used as a "time machine," should have broad application in testing models derived from evolutionary studies.

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.