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L Rowen

Publications and source records attributed to L Rowen.

29 records · Page 2Linked to original sources
PubMed1995

Frequency and polymorphism of simple sequence repeats in a contiguous 685-kb DNA sequence containing the human T-cell receptor beta-chain gene complex.

The human T-cell receptor beta-chain (TCRB) gene complex spans 575 kb in chromosome region 7q35 and has been the subject of a large-scale DNA sequencing effort. A contiguous 685-kb DNA sequence from this region was searched by computer analysis for the occurrence of simple sequence repeats (microsatellites) with core sequence lengths of 2-5 nucleotides. Twenty-nine such microsatellites of repeat number n > or = 9 were found, with the majority being dinucleotide repeats. By PCR analysis, 19 were found to be polymorphic in repeat number, thus averaging one per 36 kb. These polymorphic di-, tri-, and tetranucleotide repeats had between 3 and 15 differently sized alleles each. The potential usefulness of these TCRB microsatellites for detecting disease susceptibility alleles was examined by measuring the linkage disequilibrium between these markers and flanking biallelic mutations. All but 4 microsatellites (79%) demonstrated significant linkage disequilibrium (P < 0.0001). This present study highlights the utility and potential outcomes of large-scale DNA sequencing for the identification of polymorphic simple sequence repeats.

Alleles↗
PubMed1994

The human T-cell receptor TCRAC/TCRDC (C alpha/C delta) region: organization, sequence, and evolution of 97.6 kb of DNA.

We sequenced and analyzed 97.6 kb of new DNA sequence containing the human TCRAC (C alpha) and TCRDC (C delta) genes as well as the TCRDV3 (V delta 3) and 61 different TCRAJ (J alpha) gene segments and compared its organization and structure to the previously described mouse T-cell receptor TCRAC/TCRDC (C alpha/C delta) region. A comprehensive nomenclature, consistent with the IUIS nomenclature committee recommendations, for both human and mouse TCRAJ gene segments is presented. In the human sequence, we identified 20 new TCRAJ gene segments and obtained the germline sequence for 23 additional TCRAJ gene segments known from cDNA clones. Using the sequence data obtained from the human TCRAC/TCRDC region, we have extended a polymerase chain reaction-based assay to test for the expression of the individual TCRAJ gene segments. At least five TCRAJ pseudogene segments were identified by sequence criteria. Like the murine TCRAC/TCRDC sequence, this sequence contains a high level of coding sequence, with over 6.6% of the total sequence being transcribed. Comparison of the human sequence with the previously reported mouse DNA sequence reveals homologous counterparts for the variable and joining (J) gene segments and both constant genes. Eleven new J pseudogene segments have been identified in the mouse TCRAC/TCRDC sequence through the use of human and mouse sequence comparisons. In terms of structure and organization, this region of the human and mouse genome appears to be remarkably conserved.

Amino Acid Sequence↗
PubMed1982

Cloning of bacterial DNA replication genes in bacteriophage lambda.

Recombinant lambda phages containing the genes for dnaZ protein (the gamma subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichia coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.

Bacterial Proteins↗
PubMed1979

Gene for the RNA polymerase sigma subunit mapped in Salmonella typhimurium and Escherichia coli by cloning and deletion.

The genes for the RNA polymerase sigma subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into lambda vectors. Combined restriction, deletion and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into lambda, contains rpoD and at least part of dnaG.

Bacteriophage lambda↗
PubMed1978

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.

Coliphages↗
PubMed1978

Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains.

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase.

Coliphages↗
PubMed1978

The RNA primer synthesized by primase to initiate phage G4 DNA replication.

With phage G4 DNA as template, primase (the dnaG protein) synthesizes a 26- to 29-residue RNA transcript at the origin of replication. The sequence starts with ATP and contains a hairpin region of one A-U and seven G-C base pairs. Covalent linkage between the RNA and the newly synthesized complementary DNA chain indicates that the RNA transcript serves as a primer.

Base Sequence↗
PubMed1978

A ribo-deoxyribonucleotide primer synthesized by primase.

The 29-residue ribonucleotide primer formed by primase at the origin of phage G4 DNA replication (Bouché, J.-P, Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 765-769) was shorter in the presence of deoxynucleoside triphosphates (dNTPs). At 50 micrometer dNTPs and 20 micrometer rNTPs, RNA trancripts no longer than 6 residues were synthesized and these were still effective in priming replication by the DNA polymerase III holoenzyme. Primer synthesis was initiated with ATP; adenosine 5'-O-(3-thiotriphosphate) (Appp(S)), adenosine 5'-tetraphosphate, adenylyl imidodiphosphate (App(NH)p), and ADP were able to substitute for ATP. dATP and GTP were ineffective in initiating replication. DNA replication was stimulated by GTP, suggesting that incorporation of this nucleotide into the second position of the primer trancript by primase produces a more efficient primer. Each of the dNTPs can be incorporated into a hybrid ribonucleotide-deoxyribonucleotide transcript, indicating that primase is able to add either a ribonucleotide or deoxyribonucleotide to the 3'-OH of either of ribo residue or a deoxy residue of the primer terminus. Incorporation of an individual dNTP was less efficient than that of the corresponding rNTP, and the presence of all four dNTPs profoundly depressed RNA synthesis by primase.

Adenosine Triphosphate↗