The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.     

Genetic Variation in Amount of Salivary Amylase in the Bank Vole, CLETHRIONOMYS GLAREOLA

Several investigated bank vole populations are polymorphis for the number of salivary amylase loci, and individual chromosomes may carry one, two or three linked amylase structural genes. In the present study, we have used bank vole stocks homozygous for different chromosomes to investigate the relationship between amylase production and gene number. By measuring the amylase activity in parotid glands and the percentage of amylase protein in saliva, we have been able to demonstrate that the amount of salivary amylase is directly proportional to the proposed gene number. The paper also describes the allele, AmySu, which codes for a heat-labile salivary amylase. The relative amounts of the heat-labile isozyme have been determined in different heterozygotes containing this allele, and these results also support the multiple locus model. Finally, a stock devoid of salivary amylase activity was established. Animals from this strain have, however, a protein in the parotid glands and in saliva that is very similar to amylase in molecular weight, amino acid composition and in its binding to glycogen and cyclohepta-amylose. In genetic crosses, the protein segregates as an amylase allele. Therefore, this protein, encoded by the functionally null allele AmyN, may represent an incorrectly processed amylase precursor.     

Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.     

Expression of the pfl gene and resulting metabolite flux distribution in nuo and ackA-pta E. coli mutant strains

Our laboratory previously studied the interaction between nuo and the acetate-producing pathway encoded by ackA-pta in Escherichia coli. We examined metabolic patterns, particularly the ethanol and acetate production rates, of several mutant strains grown under anaerobic growth conditions. Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formate production under anaerobic conditions, we examined the effects of nuo and ackA/pta mutations on the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. The ackA-pta mutant has a pfl::lacZ expression level much higher than that of the wild-type strain, and cultures also exhibit the highest ethanol production. Real-time PCR demonstrated that the adhE gene expression in the ack-pta mutant strain was approximately 100 fold that of the same gene in the ackA-pta nuo mutant strain. This result correlates with the observed ethanol production rates in cultures of the strain. However, the lack of exact correlation between the ethanol production rates and the RT-PCR data suggests additional regulation actions at the posttranslation level. In addition, the activity of the pfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions of nuo and ack/pta can partially affect the expression of the genes encoding adhE and pfl under anaerobic conditions.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway.     

Pearl millet, a source of alpha amylase production by Bacillus licheniformis

The present study is concerned with the selection of economically available agricultural starchy substrate for the production of alpha amylase by Bacillus licheniformis. Different agricultural starchy substrates such as soluble starch, hordium, pearl millet, rice, corn, gram and wheat starch were tested for the production of alpha amylase by parental and its mutant derivatives. The production of alpha amylase was 10-folds better by the mutant strain B. licheniformis GCUCM-30 than the parental strain when pearl millet starch at 1.5% level and nutrient broth concentrations at the level of 0.25% was supplemented to the fermentation medium.     

Interstrain Variation in Amylase Gene Copy Number and mRNA Abundance in Three Mouse Tissues

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.     

Iron toxicity protection by truncated Ras2 GTPase in yeast strain lacking frataxin

Yeast strain deleted for the YFH1 gene, which encodes the orthologue of human frataxin, accumulates iron in mitochondria, constitutively activates the high-affinity iron import system in the plasma membrane, and is sensitive to high iron media. We have performed a genetic screen for mutants of a yfh1 deleted strain with increased resistance to high levels of iron. One of the identified mutations caused the deletion of the hypervariable C-terminal region of Ras2p GTPase. The effect of ras2 mutation on the growth of yfh1 null strain was masked by the addition of caffeine. We found that the ras2 mutation does not alter the expression of the iron regulon nor prevent mitochondrial iron accumulation in a yfh1 mutant context. The double yfh1 ras2 mutant has increased mRNA levels of CIT2 gene and augmented catalase activity.     

The Candida albicans UBI3 gene encoding a hybrid ubiquitin fusion protein involved in ribosome biogenesis is essential for growth

We have constructed a conditional null mutant Candida albicans strain for the UBI3 gene which encodes a ubiquitin fusion protein involved in ribosome biogenesis. A one-step gene disruption procedure, using the plasmid pCaDis, was designed to place the second copy of the UBI3 gene under the control of the tightly regulated MET3 promoter in a C. albicans heterozygous strain (UBI3/Deltaubi3::hisG), previously isolated in the first step of the ura-blaster protocol. Analysis of the conditional null mutant in repressing and inducing conditions indicates that UBI3 is an essential gene whose expression is required for growth of C. albicans.     

Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant.

The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed.     

Two Drosophila learning mutants, dunce and rutabaga, provide evidence of a maternal role for cAMP on embryogenesis

The dunce gene of Drosophila melanogaster encodes a cAMP-specific phosphodiesterase (form II). Mutant dunce flies have elevated levels of cAMP and exhibit a number of defects including learning deficiencies and female sterility. Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells.     

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.     

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.