Changes in gene expression elicited by amino acid limitation in Neurospora crassa strains having normal or mutant cross-pathway amino acid control

Summary The effects of amino acid limitation on gene expression have been investigated in Neurospora crassa strains carrying normal (cpc-1 ⁺) or mutant (cpc-1) alleles at a locus implicated in cross-pathway amino acid control. Electrophoresis and fluorography were used to reveal the patterns of label incorporation into polypeptides in vivo, or after in vitro translation of extracted mRNAs. In a cpc-1 ⁺ strain at least 20% of detectable in vitro translation products showed relative increases in incorporation when RNA was obtained from mycelium grown under conditions of arginine limitation, by comparison with conditions of arginine sufficiency. A cpc-1 mutation, which impairs derepression of a variety of amino acid synthetic enzymes following amino acid limitation, had little detectable effect on in vivo polypeptide synthesis during amino acid sufficient growth or following pyrimidine limitation. However the mutation substantially altered the response to arginine or histidine limitation. The majority of in vitro translation products that showed increased expression in arginine limited cpc-1 ⁺ failed to increase in cpc-1 strains, but arginine limitation of cpc-1 also resulted in increases that did not occur in cpc-1 ⁺ strains. This may reflect both direct and indirect consequences of the impairment of cross-pathway control.     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.     

Unstable alleles of the singed locus in Drosophila melanogaster with reference to a transposon marked with a visible mutation

Summary The gene clw can be integrated into another site in the X chromosome to form the mutable two gene transposon system sn::Tn-clw. When brought under the common control of the transposon, sn and clw can concomitantly mutate and manifest themselves. Tn-clw was found to move from the sn region to a position of±52 m.u. This transposition was associated with changes in sn::Tn-clw:(1) loss of the instability property by the sn ⁺ allele; (2) appearance of a new lethal allele, clw ⁹, designated as a transposon allele. Based on analysis of the direction and frequency of the sn-clw mutations, the unstable genes of the sn locus were grouped in three pleiades. Interallelic mutations occurred regularly at frequencies of 0.1%–5.6% with changes in the manifestation of the clw mutation specific to each pleiade. Transition from one pleiade to another was rare (5×10^–4), and it was associated with a new phenotypic expression of the sn and clw alleles. The mutational differences between the pleiades are presumably related to differences in the localization of Tn-clw within the sn locus.It was shown that the presence of Tn promotes recombination events in the rightmost portions of the sn-oc interval.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Facilitated isolation of rare recombinants by ligase chain reaction: selection for intragenic crossover events in the Drosophila optomotor-blind gene

Ligase chain reaction (LCR) was evaluated as a tool for the detection of point mutations. For the mutation studied, the specificity of the method is sufficient to detect the mutant allele in the presence of a 200-fold molar excess of the wild-type sequence. LCR was therefore employed in a genetic recombination experiment as a probe for a recessive lethal point mutation. LCR greatly facilitated the isolation of a rare recombinant originating from a crossover event in the 40 kb interval separating the lethal mutation and an enhancer trap insertion in the optomotor-blind locus. The recombinant will allow the study of gene control in situ, in a largely unperturbed regulatory environment.     

Identification of alleles at the gliadin loci Gli-U1 and Gli-Mb1 in Aegilops biuncialis Vis.

The diversity of alleles at the gliadin loci Gli-U1 and Gli-M ^b 1 was studied in the tetraploid species Aegilops biuncialis (UUM^bM^b). The collection of 41 Ae. biuncialis accessions and F₂ seeds obtained from five crosses served as the material used in this study. Gliadins were separated by acid polyacrylamide gel electrophoresis. To determine genomic affiliation (U or M^b) of components of Ae. biuncialis gliadin pattern, accessions of Ae. umbellulata and Ae. comosa were analyzed. In Ae. biuncialis accessions, 14 alleles were identified at the locus Gli-U1 and 12 alleles, at the locus Gli-M ^b 1. The results testify to a high degree of allele diversity at major gliadin-coding loci of homeologous group 1 chromosomes of Ae. biuncialis.     

The dominant Drop eye mutations of Drosophila melanogaster define two loci implicated in normal eye development

The three existing dominant gain-of-function Drop alleles, Dr ¹, Dr ^Mio and Dr ^We , previously assumed to define a single locus, severely disrupt eye development. Genetic analysis of ethylmethanesulphonate (EMS) and irradiation-induced revertants revealed that the Drop mutations define two loci: the Drop locus, which is defined by the Dr ¹ and Dr ^Mio mutants, and a separate locus defined by the Dr ^We mutation, which has been renamed Wedge. The majority of the Dr ¹ and Dr ^Mio revertants are embryonic lethal in trans, mutant embryos exhibiting trachea that fail to join the Filzkörper, thus revealing a role for the Drop gene in embryogenesis. Clonal analysis of lethal revertant alleles suggests a role for both genes in eye development. In the Drop homozygous mutant clones, the outer photoreceptor cells R1–R6 develop aberrantly. Wedge, however, is not required by the developing photoreceptor cells but its absence does disrupt normal ommatidial alignment. Although the Drop and nearby string loci were shown to be genetically distinct, both Dr ¹ and Dr ^Mio were found to interact in trans with lesions at the string locus, causing loss and derangement of bristles and loss of neuromuscular coordination.     

cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle.

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.     

Spm and I element changes with the a-m2 8004 allele in maize

Summary Among the mobile element systems in maize, the En (Spm) system (En — the regulatory element and I the receptive element — a nonfunctional En) has several interesting aspects of control of gene expression (En and Spm are homologous in structure and activity). One of the alleles arising from the Spm group included the a-m2 8004 allele that has a low spotting pattern and unique ringed areas. The interest in this allele is that Spm or En will induce it to co-express the A phenotype and mutability. Several exceptions of the allele were analyzed. Two are Spm changes and two are I changes. The analysis shows that the heritable changes include I changes that are co-expressed in various grades of color and different degrees of mutability. All these changes occur with I at the locus. The Spm changes also include changes in mutability patterns and a mottling pattern.Journal Paper No., J-11792 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

Zn++ stimulation of the induction of alcohol dehydrogenase by anaerobiosis in roots of Zea mays L.

Summary Zn⁺⁺ at an optimum concentration of 5×10^–4 M caused a two fold stimulation in the level of alcohol dehydrogenase (ADH) induced by anaerobic conditions. Isozymes specified by different genes and alleles show disproportionate increases in activity, such that, unequal representation of gene products while not eliminated, is invariably reduced by Zn⁺⁺ treatment. Thus in the case of alleles at the Adh-1 locus, there was a greater increase in the protein subunit specified by the Adh-1S allele. From previous work (Fischer and Schwartz, 1973) this protein is known to have a reduced affinity for Zn⁺⁺. This suggests that zinc availability during ADH induction is limiting and may provide an alternative to the cis-linked regulatory gene model proposed by Schwartz (1971) to explain the unequal expression of genes and alleles.     

Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE

Summary A nif regulatory gene in R. leguminosarum PRE was identified by interspecies DNA hybridization and site-directed Tn5 mutagenesis. Significant homology was found with the K. pneumoniae nifA locus, a R. meliloti symbiotic regulatory gene and E. coli ntrC; Tn5 insertions within this nifA gene inhibit the expression of the nifHDK operon, encoding synthesis of the nitrogenase polypeptides.Specific DNA hybridization also was detected between a downstream adjacent part of the PRE sym plasmid and the R. leguminosarum 248 fixZ gene, a homologue of the K. pneumoniae nifB locus. To detect further fix genes we investigated a region of the sym plasmid which is localized within a short distance upstream from the nifA gene and is transcribed selectively at a high rate during symbiosis. This approach revealed the existence of a fix cluster in which Tn5-mutations cause a Fix^- phenotype although wild-type levels of nitrogenase synthesis were detectable. In a sym plasmid fragment, which is immediately upstream adjacent to the nifA locus and only moderately expressed in Rhizobium bacteroids, a second fix gene conferring the same symbiotic phenotype was detected.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.