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M D Adams

Publications and source records attributed to M D Adams.

At least 73 records · Page 4Linked to original sources
PubMed1994

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Expressed sequence tags identify a human isolog of the suil translation initiation factor.

The complete cDNA sequence of a human isolog of the yeast suil translation initiation factor gene was obtained by assembling over 40 expressed sequence tags (ESTs) for this gene obtained from a variety of tissue-specific cDNA libraries. The human suilisol gene product is a 113 amino-acid polypeptide similar to proteins known from yeast, rice, mosquito, and Methanococcus. The identification of suilisol illustrates the utility of assemblies of independent ESTs for deriving full-length cDNA sequences for new human genes.

Amino Acid Sequence↗
PubMed1993

Unconventional processing of the 3' termini of the Epstein-Barr virus DNA polymerase mRNA.

Northern blot analysis of the Epstein-Barr virus DNA polymerase mRNA identified two discrete sizes of virally encoded polymerase transcripts, 5.08 kb detected in strains P3HR1, Raji, W-91, and FF-41 and 3.7 kb detected solely in the prototype B95-8 strain. 3' S1-nuclease mapping and analysis of cDNA sequence generated by RNA-based PCR demonstrated that the 3.7-kb polymerase mRNA from B95-8 terminates 484 base pairs downstream of the open reading frame in a region of the genome remarkable for its lack of an apparent polyadenylylation signal. Moreover, between the cleavage point and the poly(A) tract of the cDNAs are a series of inserted nucleotides, mostly adenosine and uridine residues of unknown origin. A similar analysis of the 3' terminus of the 5.0-kb mRNA from the other cell lines revealed that polyadenylylation occurs 1.4 kb downstream of the B95-8 terminus. This region is deleted in B95-8, which accounts for the alternate upstream terminus used in B95-8. Like the 3.7-kb terminus, the 5.0-kb terminus lacks a canonical polyadenylylation signal, but contains a rarely used UAUAAA sequence 32 bp upstream of the poly(A) tail. These results indicate that the mRNA encoded by the Epstein-Barr virus DNA polymerase gene is polyadenylylated at two different termini without the use of canonical signals, raising the possibility of involvement of a virus-encoded factor in 3' processing of this message.

Animals↗
PubMed1993

3,400 new expressed sequence tags identify diversity of transcripts in human brain.

We present the results of the partial sequencing of over 3,400 expressed sequence tags (ESTs) from human brain cDNA clones, which increases the number of distinct genes expressed in the brain, that are represented by ESTs, to about 6,000. By choosing clones in an unbiased manner, it is possible to construct a profile of the transcriptional activity of the brain at different stages. Proteins that comprise the cytoskeleton are the most abundant; however, a large variety of regulatory proteins are also seen. About half of the ESTs predicted to contain a protein-coding region have no matches in the public peptide databases and may represent new gene families.

Amino Acid Sequence↗
PubMed1993

Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Amino Acid Sequence↗
PubMed1992

Sequence identification of 2,375 human brain genes.

We recently described a new approach for the rapid characterization of expressed genes by partial DNA sequencing to generate 'expressed sequence tags'. From a set of 600 human brain complementary DNA clones, 348 were informative nuclear-encoded messenger RNAs. We have now partially sequenced 2,672 new, independent cDNA clones isolated from four human brain cDNA libraries to generate 2,375 expressed sequence tags to nuclear-encoded genes. These sequences, together with 348 brain expressed sequence tags from our previous study, comprise more than 2,500 new human genes and 870,769 base pairs of DNA sequence. These data represent an approximate doubling of the number of human genes identified by DNA sequencing and may represent as many as 5% of the genes in the human genome.

Brain Chemistry↗
PubMed1992

Chromosomal assignment of 46 brain cDNAs.

Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mapping of 46 brain ESTs, as well as observations on the amplification of rodent sequences.

Animals↗
PubMed1992

Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.

A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.

Amino Acid Sequence↗
PubMed1992

Regulation of the Epstein-Barr virus DNA polymerase gene.

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.

Amino Acid Sequence↗
PubMed1992

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Amino Acid Sequence↗
PubMed1991

Complementary DNA sequencing: expressed sequence tags and human genome project.

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

Amino Acid Sequence↗
PubMed1991

Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli.

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.

Bacterial Proteins↗
PubMed1991

Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli.

The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli.

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.

Amino Acid Sequence↗