PubMed Health⌕ Search

Biomedical subjects

D R Tolan

Publications and source records attributed to D R Tolan.

48 records · Page 3Linked to original sources
PubMed1987

The hisT-purF region of the Escherichia coli K-12 chromosome. Identification of additional genes of the hisT and purF operons.

A 9.7-kilobase pair segment of the Escherichia coli chromosome spanning the hisT and purF loci has been characterized. Six genes were identified in this region by complete DNA sequence analysis, in vivo expression in maxicells, and RNA transcript analysis. S1 nuclease analysis has demonstrated that some of these genes are part of the hisT or purF operons. Two of the newly identified genes, dedA and dedB, were localized immediately downstream of hisT in the hisT operon. Two other genes, denoted dedC and dedD, have been localized between the hisT and purF operons. The other two genes, dedE and dedF flank the purF gene. dedE has been previously described as the first gene in the purF operon (Makaroff, C.A., and Zalkin, H. (1985) J. Biol. Chem. 260, 10378-10387). dedF was localized downstream from purF and is part of the purF operon. In addition, dedF is homologous to the ubiX gene of Salmonella typhimurium. Adjacent to dedF is the E. coli homologue of the S. typhimurium argT locus encoding the lysine/arginine/ornithine-binding protein. All of the genes in this region of the chromosome were found to be transcribed in a counter-clockwise direction on the E. coli map which revises the direction of purF transcription.

Base Sequence↗
PubMed1987

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones.

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.

Amino Acid Sequence↗
PubMed1987

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.

Base Sequence↗
PubMed1986

The complete amino Acid sequence for the anaerobically induced aldolase from maize derived from cDNA clones.

A cDNA library was synthesized from maize anaerobic root mRNA and screened with cDNA specific to the anaerobically induced Zea mays cytoplasmic aldolase. At least 1% of the cDNA of the library corresponded to maize cytoplasmic aldolase. The sequence of four overlapping cDNA clones encoded a protein of molecular weight 38,611 homologous to aldolase. These cDNAs were polymorphic at three bases and one of these cDNAs had a different, shorter 3'-untranslated region. No known eukaryotic poly(A) addition site was detected. The derived amino acid sequences of maize was compared to the sequence of aldolase of trypanosome, Drosophila, and two mammalian isozymes, A and B. Of these, maize cytoplasmic aldolase was found to have the highest homology (55%) with rabbit aldolase A.

Journal Article↗
PubMed1986

Characterization of the human aldolase B gene.

The structure of the human gene encoding the aldolase B isozyme has been determined, including the sequence of 14,887 base-pairs. The 5'- and 3'-ends have been determined by S1 mapping. There is a single gene for this enzyme in humans that was determined from the sequence and restriction enzyme digestions of genomic DNA. The gene is 14,500 base-pairs long containing nine exons. In addition, 924 and 208 base-pairs of the 5'- and 3'-flanking region, respectively, have been determined. There is a high degree of conservation of nucleic acid sequence between aldolase B genes of human, rat and chicken. The conservation extends to untranslated and flanking regions, and includes the derived protein structures. In the 5'-flanking region there are several sequence elements that are conserved in vertebrate aldolase B genes in addition to the T-A-T-A and C-C-A-A-T boxes. These sequences may be involved in the co-ordinate and tissue-specific control of expression of this gene. Several possible polymorphic sites were detected in the sequence of the human gene which may be useful for linkage mapping of the human genome and diagnostic analysis of alleles in families with hereditary fructose intolerance.

Animals↗
PubMed1985

Spot-blot analysis of sorted chromosomes assigns a fructose intolerance disease locus to chromosome 9.

The aldolase B gene was mapped to chromosome 9 using a rapid gene mapping system. This system uses a dual-laser sorter to identify and separate metaphase human chromosomes stained with either DIPI-chromomycin or Hoechst-chromomycin. Chromosome panels were constructed from a normal cell line by sorting 22 chromosome fractions directly onto nitrocellulose filters. Twelve labeled gene probes hybridized to the sorted chromosomal DNA fractions predicted by previous chromosome assignments. Eighteen newly cloned genes have been mapped using the same protocol.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1984

The complete nucleotide sequence for rabbit muscle aldolase A messenger RNA.

The complete nucleotide sequence of rabbit muscle aldolase mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 1375 nucleotides, exclusive of the poly(A) tails. The 5'-untranslated region contains 62 bases including a potential ribosome-binding site. The 3'-untranslated region is 221 bases long. The remaining 1092 nucleotides are in an open reading frame coding for 364 amino acids. There is a single methionine residue preceding the NH2-terminal proline. The deduced amino acid sequence corrects a number of discrepancies between published structures as well as assignments previously missed. The sequences of the cloned cDNAs suggests there may be microheterogeneity in the messenger RNA population.

Amino Acid Sequence↗
PubMed1984

Complete amino acid sequence for human aldolase B derived from cDNA and genomic clones.

Several aldolase B clones from a human liver cDNA library have been identified by using a rabbit aldolase A cDNA as a hybridization probe. The most complete of these, pHL413, is 1389 base pairs long and covers approximately equal to 80% of the length of the mRNA, including 90% of the translated region. The cDNA, pHL413, was used to identify a genomic clone, lambda HG313, which encoded the remaining amino acids of human aldolase B. We demonstrate that the amino acid and nucleotide sequences of aldolase are strongly conserved even between different isozymes. Furthermore, in the 3'-untranslated regions of the mRNAs for the B isozyme of human and rat there is an extensive stretch of homology. Aldolase B lacks a cysteine at positions 72 and 338 and lacks a histidine at position 361. These residues, which are present in rabbit aldolase A, have previously been proposed to take part in catalysis. Our findings suggest that this may not be the case.

Amino Acid Sequence↗
PubMed1983

Crosslinking of eukaryotic initiation factor eIF3 to the 40S ribosomal subunit from rabbit reticulocytes.

Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.

Animals↗
PubMed1981

Protein topography of the 40 S ribosomal subunit from rabbit reticulocytes shown by cross-linking with 2-iminothiolane.

The small ribosomal subunit from rabbit reticulocytes was allowed to react with 2-iminothiolane under conditions that minimize the formation of 40 S subunit dimers. Reaction with 2-iminothiolane results in the formation of amidine-linked sulfhydryl derivatives of protein amino groups. Cross-linking between proximal sulfhydryl groups was promoted by mild oxidation of the modified ribosomal subunits. Protein extracted from cross-linked ribosomes was fractionated on the basis of charge by polyacrylamide-urea gel electrophoresis at pH 5.5. Cross-linked protein dimers in sequential slices of this gel were analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Constituent proteins of cross-linked pairs were located by their common mobility in the first dimension and by comparison of the sum of their molecular weights with that of the parent cross-linked species. They were unambiguously identified by extraction, radioiodination, two-dimensional polyacrylamide-urea gel electrophoresis and radioautography and comparison with nonradioactive markers. Thirty-six protein dimers were identified. Many proteins were found to occur in several cross-linked dimers and this facilitated representation of the results in a model showing the network of crosslinks. The results are discussed in relation to other structural and functional data on the 40 S ribosomal subunit.

Animals↗
PubMed1981

The purification and characterization of multiple forms of protein synthesis eukaryotic initiation factors 2, 3, and 5 from rabbit reticulocytes.

Eukaryotic initiation factors 2, 3, and 5 (eIF-2, eIF-3, and eIF-5) each were purified or fractionated into a number of forms which differ in protein mass or composition. The various factor polypeptides were analyzed by partial protease digestion and the fragmentation patterns were compared to identify proteins related in primary structure. The digestion method was made more sensitive by prior radiochemical labeling of the proteins by iodination with 125I. At least four molecular weight forms of eIF-5 were found which ranged from 168,000 to 128,000. The lower molecular weight forms, nevertheless, were biologically active in in vitro assays. Analysis of a variety of eIF-3 preparations indicates that the presence or abundance of especially the higher molecular weight subunits varies considerably. Many of these components are related to one another in primary structure; five of the subunits greater than Mr = 90,000 appear to be derived from the 210 kilodalton subunit by limited proteolysis. Analysis of eIF-2 polypeptides shows that the alpha, beta, and gamma subunits give distinctively different partial protease digestion patterns and that some preparations contain a minor component (gamma') related to the gamma subunit which could be misidentified as the beta subunit. The results indicate that initiation factors may be cleaved into different active forms by proteases acting either in the intact cell or during the factor isolation and purification. Addition of the protease inhibitor, phenylmethanesulfonyl fluoride, prevents some, but not all of the proteolysis.

Animals↗