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Biomedical subjects

J Hindley

Publications and source records attributed to J Hindley.

At least 37 records · Page 2Linked to original sources

Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe.

Sucl+ was originally identified as a DNA sequence that, at high copy number, rescued Schizosaccharomyces pombe strains carrying certain temperature-sensitive alleles of the cdc2 cell cycle control gene. We determined the nucleotide sequence of a 1,083-base-pair Sucl+ DNA fragment and S1 mapped its 866-nucleotide RNA transcript. The protein-coding sequence of the gene is interrupted by two intervening sequences of 115 and 51 base pairs. The predicted translational product of the gene is a protein of 13 kilodaltons. A chromosomal gene disruption of Sucl+ was constructed in a diploid S. pombe strain. Germinating spores carrying a null allele of the gene were capable of very limited cell division, following which many cells became highly elongated. The Sucl+ gene was also strongly overexpressed under the control of a heterologous S. pombe promoter. Overexpression of Sucl+ is not lethal but causes a division delay such that cells are approximately twice the normal length at division. These data suggest that Sucl+ encodes a protein which plays a direct role in the cell division cycle of S. pombe.

Alleles↗

Human Sau3A repeated DNA is enriched in small polydisperse circular DNA from normal lymphocytes.

Representatives of the Sau3A family of short human repeated sequences [Meneveri et al., J. Mol. Biol. 186 (1985) 483-489] have been isolated from the small polydisperse circular DNA (spcDNA) of peripheral human lymphocytes. The prototype repeat is a 72-bp element which is at least partially tandemly repeated in spcDNA and human genomic DNA. In comparison with three major families of human repeated DNA, the Sau3A repeats are enriched in spcDNA. The function of spcDNA in normal and transformed eukaryotic cells is not understood and most studies have attempted to resolve this problem by molecular analysis of circular DNA isolated from cells in culture [see Rush and Misra, Plasmid 14 (1985) 177-191 for references]. We have studied the spcDNA present in normal uncultured human lymphocytes and present data pointing to the selective accumulation of the Sau3A family of repeated DNA within this population. The sequences of twelve of these repeats, the consensus sequence for this family and the sequence of a genomic repeat, are presented.

Base Sequence↗

Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases.

The complete nucleotide sequence of a 2.9-kb DNA fragment containing the CDC2 gene-complementing activity from Schizosaccharomyces pombe has been determined. Within this region lies a 1.69-kb DNA sequence whose predicted amino acid sequence shows extensive homology to that previously deduced for the CDC28 gene product from Saccharomyces cerevisiae [Lörincz and Reed, Nature 307 (1984) 183-185]. Taken with the earlier observation that mutants in CDC2 can be rescued by the presence of the CDC28 gene [Beach, Durkacz and Nurse, Nature 300 (1982) 706-709], these results strongly suggest that the two genes code for similar functions. In contrast to the CDC28 gene, however, which contains no introns, the CDC2 coding sequence is split by four introns and from a comparison of the two sequences a consensus sequence for intron splicing in S. pombe can be established. Both CDC2 and CDC28 contain the consensus sequences for the ATP binding and phosphorylation acceptor sites of protein kinases such as bovine cAMP-dependent protein kinase (bov PK) and the src family of viral oncogene products.

Amino Acid Sequence↗

Passage of bacterial DNA into host cells during in vitro transformation of Nicotiana tabacum by Agrobacterium tumefaciens.

Tumorigenic and non-tumorigenic strains of Agrobacterium tumefaciens were labelled with tritiated thymidine and added separately to suspension cultures of X-D line habituated tobacco cells. The subsequent association of bacteria with plant host cells and the passage of bacteria and bacterial DNA into these cells was examined by conventional light and electron microscopy and light microscope autoradiography. The results suggest that both strains of bacterium enter damaged host cells (via wall lesions), where they remain restricted to cell vacuoles. By 48 h, the radioactive label has spread from the vacuoles of infiltrated cells to the non-vacuolar cytoplasm of the whole callus tissue. Tumorigenic bacteria differ from the non-tumorigenic strain in promoting a significant increase in host cell nuclear volume, which is paralleled by clear and specific nuclear labelling. It is proposed that tumorigenic DNA, but not non-tumorigenic DNA, is specifically incorporated into, and activates, the host cell nucleus.

Cell Nucleus↗

Sequencing long DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin.

In the ;shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.

Base Sequence↗

Sequence of 863 nucleotides encompassing the PstI site of the yeast 2 micron plasmid.

The sequence of part of the larger unique region of the yeast 2 micron plasmid cloned in pMB9 has been determined. The sequence extends from the single EcoRI site in this region to the AvaI site and includes the single PstI site and HpaI site. A notable feature of this sequence is the presence of tandem repeats of 124 residues beginning at the HpaI site and extending beyond the AvaI site. The sequence was determined independently by both the Maxam-Gilbert procedure applied to isolated restriction fragments, and by the chain-termination procedure applied to restriction fragments cloned in the single-stranded phage M13mp2 and purified by plaque selection.

Base Sequence↗

Sequence of 1019 nucleotides encompassing one of the inverted repeats from the yeast 2 micrometer plasmid.

A sequence of 1019 nucleotides encompassing one of the 600 base inverted repeats and non-repeated flanking regions has been determined in the type A yeast 2 micrometers plasmid cloned in pMB9. Methods are described for applying the Maxam-Gilbert sequencing procedure to DNA fragments labelled at the 3'-end using a T4-polymerase exchange/repair reaction and for sequencing 5'-end labelled fragments using dideoxy-nucleotides as chain terminators in the presence of E. coli DNA polymerase (nach Klenow). A notable feature of the sequence is its unusual content of symmetry elements. In one region of 140 nucleotides, 137 are involved in a complex arrangement of direct and inverted repeats linked by palindromic sequences.

Base Sequence↗

Cloning of human satellite III DNA: different components are on different chromosomes.

Two fragments cloned from purified human satellite III DNA do not cross-react with each other. One fragment, for which a partial sequence is reported, hybridises to satellite II as well as III and is shown to originate on chromosome 1. The other cloned fragment originates from the Y chromosome. This fragment has undergone considerable changes in size when cloned in lambda gt WES lambda B. Human satellite III is shown to consist of a number of non-cross-reacting sequences which nevertheless are related by the presence of closely spaced Hin F1 sites.

Animals↗

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.

DNA Restriction Enzymes↗