Molecular cloning of the RAD10 gene of Saccharomyces cerevisiae

We have cloned the RAD10 gene of Saccharomyces cerevisiae and physically mapped it to a 1.0-kb DNA fragment. Strains containing disruptions of the RAD10 gene were found to show enhanced UV sensitivity compared with the previously characterized rad10-1 or rad10-2 mutants. The UV sensitivity of the disruption mutant is comparable to the highly UV sensitive rad1-19, rad2-delta, and rad3-2 mutants.     

Molecular cloning of the gene for dihydrofolate reductase from Lactobacillus casei

The Lactobacillus casei gene for dihydrofolate reductase has been cloned in Escherichia coli using the multicopy vector pBR322. A restriction map of the cloned DNA has been prepared. The cloned DNA directs the synthesis of L. casei dihydrofolate reductase in E. coli and confers trimethoprim and methotrexate resistance.     

Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli

The thyA gene of Escherichia coli, which directs the synthesis of the enzyme thymidylate synthetase, has been subcloned from a recombinant λ phage (Hickson et al., 1982) into the multicopy plasmid pBR325 to give the plasmid pPE245. To identify the thyA gene product, the transposon Tn1000 was inserted into pPE245 and derivative plasmids isolated that were no longer able to complement thyA mutations. When proteins synthesised by these plasmids and by pPE245 were labelled and analysed on SDS-polyacrylamide gels a protein of 33000 M_r, presumably the thyA⁺ gene product was absent whenever the thyA gene was inactivated. On assaying cell extracts prepared from cells harbouring pPE245 for thymidylate synthetase, the level of this enzyme was found to be elevated by a factor of at least 25.     

The yeast cloning vector YEp13 contains a tRNA3Leu gene that can mutate to an amber suppressor

We have shown that the yeast-Escherichia coli shuttle vector YEp 13 contains, as part of its yeast chromosomal segment, a tRNA₃^Leu gene. We have also isolated and characterized a variant of YEp13, namely YEp13-a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13-a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNA₃^Leu anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNA₃^Leu gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNA₃^Leu gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53. We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNA₃^Leu genes has revealed considerable sequence homology in the immediate 5′-flanking regions of these genes.     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Construction and characterization of new cloning vehicles. VII. Construction of plasmid pBR327par, a completely sequenced, stable derivative of pBR327 containing the par locus of pSC101

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.     

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a λgt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of M_r 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.