Apocynin activity in spontaneously hypertensive rats (SHR): preliminary studies in vivo.

An elevation in angiotensin II (Ang II) levels is a common occurrence in spontaneously hypertensive rats (SHRs). Infusions of Ang II and a high salt diet increase the activity of NADPH oxidase that stimulates superoxide anion (O(-2)) generation and increases the expression of certain subunits of NADPH oxidase. Apocynin, an NADPH oxidase inhibitor with antihypertensive effects, is able to inhibit the release of superoxide anion by inhibiting NADPH oxidase activity and blocking the migration of p47 phox to the mitochondrial membrane. The aim of our study was to evaluate the antihypertensive effects of apocynin in SHRs and Wistar rats (WKYs) using a micropuncture technique. After microperfusion of both the proximal and distal tubules, we found that SHRs treated with apocynin showed a decrease in the free-flow collection of the proximal tubule (PT), which was not affected in WKYs. Moreover, significant differences were not demonstrated in the distal tubule (DT), probably due a mechanism of compensation that occurs in the loop of Henle. In conclusion, it is possible that the mechanisms of reabsorption in the PT are controlled by the interactions of O(-2) and nitric oxide (NO). These data could suggest a higher activity of NADPH oxidase and increase in reactive oxygen species (ROS) production in the PT during hypertension.

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.

Mutagenesis of yeast MW104-1B strain has identified the uncharacterized PMS6 DNA mismatch repair gene locus and additional alleles of existing PMS1, PMS2 and MSH2 genes.

The haploid yeast Saccharomyces cerevisiae MW104-1B strain was disomic for chromosome III (n + 1) and carried DNA mismatches at three different heteroallelic loci; leu2 (leu2-1/leu2-27), thr4 (thr4-1/thr4-16) and his4 (his4-4/his4-519) (Williamson, 1984). We mutagenized the MW104-1B strain and identified seven mutant isolates that display elevated mitotic/meiotic prototrophs due to mismatch repair failures at heteroallelic loci. Three mutants (pms1, pms2 and pms3) isolated earlier from MW104-1B were shown to correct in vitro constructed plasmids with defined DNA mismatches (G/T, A/C, G/G, etc.) poorly (Kramer et al., 1989a). Complementation tests were performed by crossing all seven new mutant isolates to pms1 and pms2 mutants and assaying for mutant phenotype in the diploids. Four mutant isolates failed to complement the two known pms alleles (pms1-1 and pms2-1). Two other mutant isolates complemented the pms1-1 and pms2-1 alleles, but failed to complement each other and were named as the pms5-1 allele of an uncharacterized gene (PMS5). One other mutant isolate complemented the pms1-1, pms2-1 and pms5-1 alleles and was named as the pms6-1 allele of another uncharacterized gene (PMS6). Subsequently, the pms5-1 mutant allele was shown to be complemented by a plasmid borne yeast MSH2 gene, implying that it is an allele of MSH2 (PMS5). The human homologs (hMSH2 and hMLH1) of two yeast DNA mismatch repair genes (MSH2 and MLH1) have been cloned recently and shown to be responsible for hereditary nonpolypnosis colon cancer (HNPCC) (Fishel et al., 1993; Leach et al., 1993; Bronner et al., 1994; Papadopoulos et al., 1994).

Gene conversions within the Cup1r region from heterologous crosses in Saccharomyces cerevisiae.

Meiotic recombination among unselected tetrads was analyzed genetically and physically in a heterologous cross where one parent carried six copies of a 1.6 kb CUP1 repeat while the other parent carried seven copies of a 1.1 kb repeat. In the heterologous cross, 140 unselected, complete tetrads were subjected to Southern analysis and 20% exhibited meiotic copy number alterations at the CUP1 locus. Most events, more than 75%, involved only a single spore of a tetrad, and were largely intrachromosomal or sister chromatid events. However, some conversions and associated crossovers between homologs were also observed. We propose that the high level of heterologies interferes with homologous exchanges and leads to an increase in intrachromosomal events.

Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast.

A 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02 microM) or copper (50 microM) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-3B delta, deleted for the CUP1 gene (cup 1 delta), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.

Unequal crossing-over and gene conversion at the amplified CUP1 locus of yeast.

Meiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at the CUP1 locus of Saccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.

The molecular genetics of copper resistance in Saccharomyces cerevisiae--a paradigm for non-conventional yeasts.

After a short introduction on the historical background of the development of Saccharomyces cerevisiae as a model eukaryote, a review is given on the present state of genetics and molecular biology of copper resistance in S. cerevisiae. The gene CUP1 encodes a protein (copper metallothionein) of a molecular weight of 6570 dalton. The synthesis of this copper chelatin is induced by copper and is regulated at the level of transcription. Copper resistance (CUPr) is correlated with amplification of CUP1 and resulted in a higher copy number of this gene on chromosome VIII. Spontaneous meiotic alterations of the gene copy number have been studied.

Synaptic relations in meiotic gene conversion at the iterated CUP1r locus of S. cerevisiae.

This study concerns a comparative molecular analysis of copy number changes in two hybrids that differ in the extent of homologies at the CUP1r locus. Hybrid JW1020 is a diploid wherein each parent contributed nine identical, tandemly arrayed 2.0 kb repeat units. Genomic DNA was isolated from each of the spore colonies in a sample of 200 unselected tetrads. About 15% displayed copy number changes, i.e., increases or decreases of one or more complete units. Changes on a per tetrad basis occurred as often in a single spore colony as changes in each of two spores. Such double changes are rarely reciprocal in character. To account for the observed qualitative and quantitative copy number shifts, we propose a molecular recombination model that posits partial, incomplete synaptic pairing and gene conversion of the unpaired regions with or without associated crossing over. A second contrasting study centers on the copy number alterations and recombinational events uncovered in a molecular analysis of 50 unselected tetrads generated by hybrid EB8. Unlike the hybrid JW1020, the EB8 diploid strain carried a six copy tandem array of 1.1 kb units at the CUP1r locus in one parental homologue and a five copy array of 1.6 kb units at the corresponding chromosome VIII locus. These natural alleles were recovered from industrial yeast strains by conventional genetic procedures and characterized by restrictional analysis. Twelve tetrads exhibit evidence for several different types of recombination events. However,ordinary crossover exchanges are conspicuously absent. We suppose that the repetitious nonhomologies generate DNA configurations sufficient to disrupt the effective synapsis over the entire locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Industrial yeasts display tandem gene iteration at the CUP1 region.

The gene copy number at the CUP1 locus and the resistance level to external copper was directly correlated in five wild-type commercial Saccharomyces strains. An increased copy number of the CUP1 gene leads to increased accumulation of chelatin mRNA, which codes for a low-molecular-weight, copper-binding protein. The enhanced production of this rapidly inducible protein mediates resistance of the cell to copper. Industrial yeasts exhibit homologies to the amplified copper resistance repeat unit found in laboratory strains. However, the extent of tandem iteration is strain dependent, and the repetitious unit is either 1.7 or 1.5 kilobases in length compared with the 2.0-kilobase unit in laboratory strains. Strain 522 (Montrachet) contains two chromosome VIII segments distinguishable by their numbers of repeat units (2 and 11) and the size of the units (1.5 and 1.7 kilobases). Distillers yeast 513 carries a 1.5-kilobase repeat unit on each homologous chromosome, although they contain nine and five iterations, respectively.

Gene amplification in yeast: CUP1 copy number regulates copper resistance.

The CUP1 locus in yeast confers resistance to copper toxicity. We determined the molecular basis for copper resistance in three yeast strains, with differing degrees of resistance. Increased resistance to copper is associated with overproduction of a low molecular weight copper-binding protein, copper-chelatin. Increased chelatin synthesis results from amplification of the CUP1(r) gene and increased synthesis of the copper inducible mRNA. The copper resistance level of a given strain correlates directly with the gene copy number.Strains containing one copy and ten tandemly iterated copies of the CUP1 gene were studied. From the latter, a haploid strain with enhanced resistance was isolated following several selection cycles at elevated copper concentrations. This strain was disomic for chromosome VIII, the chromosome containing the CUP1 locus. The disomic chromosomes exhibit differential CUP1 gene amplification: 11 and 14 tandemly organized repeat units are found in the respective chromosome VIII homologues. We propose that the molecular mechanisms of gene amplification involve unequal sister chromatid exchange and intrachromosomal gene conversion, as well as disomy.

Tandem gene amplification mediates copper resistance in yeast.

Resistance to copper's toxicity in yeast is controlled by the CUP1r locus. This gene was cloned by transforming sensitive recipients (cup1(8)) with a collection of hybrid DNA molecules, consisting of random yeast DNA fragments inserted into the vector YRp7. Four resistant transformants were studied in detail. Autonomously replicating or integrated by homologous recombination into chromosomal sites, the corresponding plasmids and several subclones confer resistance on sensitive recipients carrying the natural variant allele, cup1(8). Tetrad analysis and genetic mapping established that integration occurs typically at the cup1(8) site located 28 centimorgans distal to thr1, a chromosome VIII marker. Restriction endonuclease cleavage and electrophoretic mobility studies revealed that the CUP1r locus consists of a tandem array of repetitive units. Each unit is 1.95 kilobases in length and contains single sites for Kpn I and Xba I and two Sau3A sites. The sensitive allele represents one repeat and the resistant allele embraces 15 tandemly arrayed repeat units. Progressive selections in higher copper concentrations establish strains with markedly enhanced resistance. Resistance, we propose, is mediated by a gene amplification mechanism based on unequal sister chromatid exchange.

Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae.

To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.

Very long-chain fatty acids in yeast.

Novel fatty acids ranging from 20 to 30 carbons have been found in Saccharomyces cerevisiae. These comprise 1 to 2% of the total fatty acid fraction.