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Biomedical subjects

J Carbon

Publications and source records attributed to J Carbon.

At least 73 records · Page 4Linked to original sources
PubMed1980

Isolation of a yeast centromere and construction of functional small circular chromosomes.

The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.

Centromere↗
PubMed1980

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene.

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.

Aldose-Ketose Isomerases↗
PubMed1980

Isolation of the centromere-linked CDC10 gene by complementation in yeast.

A hybrid plasmid colony bank was constructed in Escherichia coli using the E. coli-Saccharomyces cerevisiae shuttle vector pLC544 and randomly sheared segments of yeast DNA. By transformation with a hybrid plasmid DNA pool from this collection and complementation of a temperature-sensitive cdc10 mutation in yeast, a plasmid was isolated that carries 8 kilobase pairs of DNA around the chromosome III centromere-linked CDC10 locus. This DNA segment overlaps a larger region of DNA (40 kilobase pairs) previously identified to be around the LEU2 locus on chromosome III [Chinault, A.C. & Carbon, J. (1979) Gene 5, 111-126] and physically establishes the directionality of the cloned DNA sequences with respect to the genetic map and the centromere. In the leu2-cdc10 interval, the relationship between physical distance on the DNA and genetic distance as measured by recombinational frequencies is about 3 kilobase pairs per centimorgan.

Cell Division↗
PubMed1979

Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region.

Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA). Transformation can be achieved either by using purified plasmid DNA or by fusion of yeast spheroplasts with partially lysed Escherichia coli [pLC544] protoplast preparations. The Trp+ yeast transformants are highly unstable, segregating Trp- cells at frequencies of 0.18 per cell per generation (haploids) and 0.056 per cell per generation (diploids) in media containing tryptophan. Plasmid pLC544 replicates autonomously in the nucleus of yeast cells and segregation of Trp-cells is associated with the complete loss of plasmid sequences. In genetic crosses, pLC544 is randomly assorted during meiosis and is carried unchanged through the mating process into haploid recombinants.

Cell Nucleus↗
PubMed1979

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.

Base Sequence↗
PubMed1979

Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III.

A set of four plasmids containing overlapping segments comprising a total of about 30 kbp of cloned DNA from chromosome III of yeast (Saccharomyces cerevisiae) has been isolated and characterized by restriction endonuclease analyses and DNA:DNA hybridizations. Colony hybridization was carried out with labeled pYe(leu2)10, a plasmid carrying the yeast leu2 gene, to a bank of bacterial colonies containing recombinant plasmids constructed from the vector ColE1 and random fragments of yeast DNA. This resulted in the detection of two plasmids, pYe11G4 and pYe40C3, with DNA inserts which partially overlap the original cloned segment and contain additional DNA extending in opposite directions on the chromosome. By carrying out a second round of colony hybridization with pYe40C3, the cloned region was further extended in one direction. A region of DNA that is repeated at least ten times in the yeast genome was identified by hybridization of pYe11G4 to an EcoRI digest of total yeast DNA. The procedure described in this paper should allow the isolation of large sections of chromosomes, including non-transcribed regions, surrounding cloned genes.

Base Sequence↗
PubMed1979

High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene.

Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4(+) with a frequency of 10(-4) (about 10(3) transformants per mug of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli "shuttle" remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.

Argininosuccinate Lyase↗
PubMed1978

Control of expression of a cloned yeast (Saccharomyces cerevisiae) gene (trp5) by a bacterial insertion element (IS2).

A hybrid ColE1 plasmid [pYe(trp5)1], containing a yeast DNA segment that complements auxotrophic point mutations and deletions in the Escherichia coli tryptophan synthetase gene (trpAB), has been isolated. Expression of the yeast tryptophan synthetase activity from the cloned yeast gene (trp5) is relatively inefficient in E. coli, as measured by growth rates of trpAB/pYe(trp5)1 strains on minimal media lacking tryptophan and by enzyme assays. Faster growing variants occur spontaneously at a frequency of one in 10(4)--10(5) cells plated and produce higher levels of the yeast enzyme. Plasmid DNA [pYe(trp5)2] from one of these variants was shown to contain a DNA insertion (1.3 kilobase pairs) in the cloned yeast DNA segment in relatively close proximity to the trp5 gene. This DNA insert was identified as a bacterial IS2 element, which carries a promoter for RNA transcription when inserted in the proper orientation. The spontaneous integration of a bacterial DNA insertion element into cloned eukaryotic DNA can result in more efficient expression of the foreign gene.

Bacteriocin Plasmids↗
PubMed1977

Nucleotide sequence of the 5' end of araBAD operon messenger RNA in Escherichia coli B/r.

The transcription reaction in vitro provides a means of analyzing the nucleotide sequence of the mRNA of the araBAD operon. By controlling the time of synthesis, we obtained araBAD mRNA of varying lengths beginning from the 5' end. These 5' fragments were freed of lambda RNA transcripts by successive hybridizations to the sense strands of a pair of lambda ara transducing phages that carry ara genes in opposite orientations. The purified 5' fragments were ordered by their times of appearance during synchronized RNA elongation and by nearest neighbor analyses. The results, when combined with the knowledge of the NH2-terminal sequence of the product of the first cistron (L-ribulokinase gene araB), establish the nucleotide sequence of the first 69 bases at the 5' end of the araBAD operon mRNA. The AUG starter codon for L-ribulokinase is located at positions 29-31. The sequence is: 5' A-C-C-C-G-U-U-U-U-U-U-U-U-G-G-A-U-G-G-A-G-U-G-A-A-A-C-G-A-U-G-G-C-G-A-U-U-G-C-A-A-U-U-G-G-C-C-U-C-G-A-U-U-U-U-G-C-A-G-U-G-A-U-U-C-U-G-(U)-. . .3'.

Arabinose↗
PubMed1977

Functional expression of cloned yeast DNA in Escherichia coli.

A collection of hybrid circular DNAs was constructed in vitro using the poly(dA-dT) "connector" method: each hybrid circle contained one molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 X 10(6) daltons. This DNA preparation was used to transform E. coli cells, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids. Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome. Various hybrid ColE1-yeast DNA plasmids capable of complementing E. coli auxotrophic mutations were selected from this population. Plasmid pYeleu 10 complements several different point or deletion mutations in the E. coli or S. typhimurium leuB gene (beta-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12, and pYeleu17 are specific suppressors of the leuB6 mutation in E. coli C600. Plasmid pYehis2 complements a deletion in the E. coli hisB gene (imidazole glycerol phosphate dehydratase). Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon.

Alleles↗
PubMed1976

A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid.

A general method has been developed for the deletion of restriction endonuclease sites in bacterial plasmid DNA. The procedure involves partial digestion of the covalently closed circular plasmid DNA with an appropriate restriction endonuclease under conditions which allow accumulation of unit-length linear DNA molecules, a controlled digestion of the exposed 5' ends with the lambda 5'-exonuclease, and in vivo recircularization of the resulting linear DNA in a bacterial host cell. The method has been used for the deletion of one of the two EcoRI sites in the plasmid pML2 (colE1-Km). Two of the resulting plasmids, pCR1 and pCR11, have a single EcoRI cleavage site, but retain genetic determinants specifying resistance to colicin E1 and kanamycin, and thus may be useful as vectors for the cloning and amplification of DNA in bacteria.

Coliphages↗
PubMed1976

Isolation and characterization of large transfer ribonucleic acid precursors from Escherichia coli.

Several transfer RNA precursors which accumulate in a strain of Escherichia coli temperature-sensitive for RNase P have been described. These precursors range from 135 to 690 nucleotides in length. Their tRNA content has been determined by digestion of the precursors to 4 S RNA, followed by Sanger fingerprint analysis of the purified 4 S material. Identification of some of these tRNAs, as well as an estimate of the number of copies of tRNA in each precursor has been achieved. Many of these precursor RNA molecules contain multiple copies of the same tRNA sequence, indicating a tandem arrangement of the corresponding tRNA genes in the E. coli genome.

Escherichia coli↗
PubMed1976

A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.

Using the poly(dA-dT) "connector" method (Lobbanand Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed Col El-DNA (LRI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 x 10(6) daltons. This annealed DNA preparation (12 mug) was used to transform an F+ recA E. coli strain (JA200), selecting transformants by their resistance to colicin El. A collection or "bank" pf pver 2000 colicin El-resistant clones was thereby obtained, 70% of which were shown to contain hybrid Col El DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F- auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F- recipients. In this manner, over 80 hybrid Col El-DNA (E. coli), plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F+ donors to appropriate F- recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.

Chromosome Mapping↗