Cloning and integrative deletion of the RAD6 gene of Saccharomyces cerevisiae

Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad⁺ levels of resistance to U.V., MMS and -rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids.Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.     

Identification of the yeast DNA polymerase I gene with antibody probes

Summary Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage expression vector gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.     

Abnormal growth induced by expression of HBsAg in the secretion pathway of S. cerevisiae pep4 mutants

The toxicity of HBsAg in the secretion pathway of pep4 strains can be progressively reduced in modified SD media containing lower concentrations of ammonium sulphate. A procedure, combining a reduction of ammonium sulphate concentration in SD media with the disruption of the PEP4 gene of the host strain, was developed to enrich transformants which are not inhibited by HBsAg expressed in the secretion pathway. Abnormal growth of these non-inhibited transformants is characterized by the enlargement of cell morphology, a transition to pseudohyphal-like growth in nitrogen-starved media, an increase in HBsAg particle production, and the enhancement of growth rate in liquid media. This suggests a new approach to overcoming the toxicity of heterologous protein in the yeast secretion pathway.     

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 ⁺ gene, which is homologous to Saccharomyces cerevisiae LYS4 ⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 ⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7 ⁺ gene with a lys2 ⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2 ⁺ gene with a his7 ⁺-marked PCR product. Strains carrying these deletions of lys2 ⁺ or his7 ⁺ serve as relatively efficient hosts for the deletion of the ade6 ⁺ gene by lys2 ⁺- or his7 ⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 ⁺ as a selectable marker, along with improved strains for the use of his7 ⁺ to mark gene deletions.     

Prolonged E55+ retrovirus expression in aged mice is associated with a decline in the anti-virus immune response

E55+ murine leukemia retrovirus (E55+ MuLV) infection of young and aged C57BL/6 (B6) mice was used to investigate the relationship between increased incidences of infection and decreased immune responsiveness of elderly individuals. Young mice decreased E55+ MuLV burden to below detectable levels by 8 weeks postinfection (p.i.). In contrast, virus burden in aged mice did not reach undetectable levels until 20 weeks p.i. A significant T cell proliferative response to E55+ MuLV was detected from 2 to 12 weeks p.i. in young mice, but was never observed in aged mice. Both age groups demonstrated significant E55+ MuLV-specific T-cell-mediated cytotoxic responses at 3 and 4 weeks p.i. and virus neutralizing antibody titers at 2, 4, 8, and 12 weeks p.i. In both cases, responses were consistently higher in young mice (P < 0.04 and P < 0.02, respectively). These results demonstrate that the observed delay in E55+ MuLV clearance by aged mice is associated with an age-related decrease in the immune response to the virus.     

The primary structure of the leul + gene of Schizosaccharomyces pombe

Summary A DNA fragment which carries the leul gene encoding beta-isopropylmalate dehydrogenase in Schizosaccharomyces pombe has been isolated by complementation of an E. coli leuB mutation. This 1.5 kb DNA fragment complements not only the S. pombe leul mutation, but also the S. cerevisiae leu2 mutation. The nucleotide sequence of the essential part of the leul gene and its flanking regions was determined. This sequence contains an open reading frame of 371 codons, from which a protein having a Mr = 39,732 can be predicted. The deduced amino acid sequence and its codon usage were compared with those of the S. cerevisiae LEU2 protein. The cloned DNA will be a useful marker when transforming S pombe.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

Cloning of the orotidine 5′-phosphate decarboxylase (ODC) gene of Schwanniomyces occidentalis by complementation of the ura3 mutation in S. cerevisiae

Summary A DNA fragment containing the gene encoding orotidine 5-phosphate decarboxylase (ODC) from the yeast, Schwanniomyces occidentalis (formerly castellii) has been isolated from a genomic library constructed in the S. cerevisiae expression vector, pYcDE8. A recombinant plasmid, p2-lA, containing a 2.47 kb insert was shown to complement the ura3-52 mutation of several strains of S. cerevisiae. This DNA insert was shown to be from Schwanniomyces occidentalis by Southern hybridization analysis. A restriction enzyme cleavage map of the insert has been derived and the ODC gene localized to a 1.1 kb region by deletion analysis. In addition, we have demonstrated that expression of ODC is not dependent on the ADHI promoter carried on pYcDE8. This is the first report of the cloning of a gene from a member of the genus Schwanniomyces.     

The human papillomavirus type 16 E5 protein alters vacuolar H(+)-ATPase function and stability in Saccharomyces cerevisiae

The human papillomavirus 16 (HPV-16) E5 oncoprotein is a small integral membrane protein that binds to the 16-kDa subunit of the vacuolar H(+)-ATPase (v-ATPase). Conservation within the family of v-ATPases prompted us to look to Saccharomyces cerevisiae as a potential model organism for E5 study. The E5 open reading frame, driven by a galactose-inducible promoter, was integrated into the yeast genome, and the resulting strain demonstrated a nearly complete growth arrest at neutral pH, consistent with defects associated with yeast v-ATPase mutants. Furthermore, this strain demonstrated a severe reduction in pH-dependent and v-ATPase-dependent vacuolar localization of fluorescent markers. Overexpression of the yeast 16-kDa subunit homolog partially suppressed E5-associated growth defects. E5 expression was correlated with a disassociation of the integral (V(o)) and peripheral (V(i)) v-ATPase sub-complexes, as well as a dramatic reduction of the steady-state levels of one mature V(o) subunit and the concomitant accumulation of its major proteolytic fragment, with unchanged levels of two V(i) subunits. Similar analyses of selected E5 mutants in yeast demonstrated a correlation between E5 biology and v-ATPase disruption. Our observations suggest that wild-type HPV-16 E5 acts during the assembly of the v-ATPase to inhibit, either directly or indirectly, V(o) stability and complex formation.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Molecular cloning of a beta-glucosidase-encoding gene from Sclerotinia sclerotiorum by expression in Escherichia coli

Summary Six Sclerotinia sclerotiorum genes previously described as being expressed specifically during the induction of cell-wall-degrading enzymes have been transferred to Escherichia coli. When tested for expression of beta-glucosidase activity using X-glu (5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside), one of of the the genes expressed a X-glu-degrading activity. The corresponding RNA size is reported.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.