Genetic Analysis of Dictyostelium Slug Phototaxis Mutants

Mapping and complementation analysis with 17 phototaxis mutations has established 11 complementation groups phoA-phoK distributed over six linkage groups. Statistical calculations from the complementation data yielded 17 as the maximum likelihood estimate of the number of pho genes assuming all loci are equally mutable. Most of the phototaxis mutants were found to exhibit bimodal phototaxis and all were found to be impaired in positive thermotaxis supporting convergence of the photosensory and thermosensory pathways. The thermotaxis mutant HPF228 was unaltered in phototaxis suggesting that the mutation in this strain affects a gene product whose site of action is before the convergence of the two pathways. Other phenotypes such as multiple tip formation by aggregates, stumpy fruiting bodies with short or absent stalks and short migration were associated with some pho alleles suggesting multiple biological roles for some gene products important in phototransduction.     

Genetics of aggregation pattern mutations in the cellular slime mould Dictyostelium discoideum.

A class of aggregation pattern mutants called 'streamers' have been isolated from Dictyostelium discoideum and analysed genetically. The streamer phenotype is the formation of very large streams of centripetally moving amoebae which are collected from abnormally large territories during the aggregation phase of this organism. Such mutants do not show the pleiotropic developmental defects seen with most other classes of aggregation mutants and after the abnormal aggregation phase they develop into normally differentiated stalk cells and spores. Twenty-four haploid streamers were isolated and assigned to seven complementation groups, stmA to stmG, after selecting diploids formed between pairs of the mutants. The complementation loci were assigned to the following linkage groups using parasexual genetic techniques: stmA and stmF, linkage group VII; stmB, stmD and stmG, linkage group II; stmC and stmE, linkage group III. Use was made of a new temperature sensitive for growth marker, tsgK21, which was assigned to linkage group VII. The total number of complementation groups giving the streamer phenotype is estimated from statistical calculation, based on the frequency of allelism, to be between seven and nine.     

Genetic Map of Bacteriophage α

Temperature-sensitive mutants of phage alpha were obtained by means of various mutagens and assigned to 25 complementation groups. Temperature-sensitive mutants belonging to 21 complementation groups and a mutant giving turbid plaques were used to perform two- and three-factor crosses. Seventeen of the cistrons and the turbid mutant were shown to belong to the same linear linkage group, which showed no signs of circularity. The remaining four unlinked cistrons showed peculiarities in their recombination properties. Genes which are known to be expressed earlier apear to be grouped together in a terminal segment of the linkage group.     

The ATC (ataxia-telangiectasia complementation group C) locus localizes to 11q22-q23.

The multisystem autosomal recessive disease ataxia-telangiectasia (A-T) is determined by several genes, as evidenced by the existence of four complementation groups in this disorder. Using linkage analysis, the ATA (A-T complementation group A) gene was previously localized to chromosome 11, region q22-q23. Analysis of the segregation of RFLP markers from this region in a Jewish-Moroccan family assigned to group C indicates that the ATC (A-T complementation group C) gene localizes to chromosome 11q22-q23 as well.     

Genetic classification of riboflavin-dependent mutants of Pichia guilliermondii yeasts]

114 riboflavinless mutants were selected from the genetic line of Pichia guilliermondii yeast. By means of accumulation test the mutants were divided into five biochemical groups. In genetic experiments seven complementation classes were found among 106 mutants. The strains of the I biochemical group, accumulating no specific products, corresponded to complementation class rib1; II group, accumulating 2,4,5-triaminopyrimidine - to the class rib2; III group, accumulating 2,6-dihydroxy-4-ribitylaminopyrimidine - to the class rib3; the mutants of the IV group, accumulating 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine, were divided into three complementation classes rib4, rib5 and rib6; the mutants of the V group, acculumating 6,7-dimethyl-8-ribityllumazine, corresponded to the class rib7. Two mutants of the IV biochemical group within complementation classes rib4 and rib5 were detected could not grow in the medium with diacetyl without riboflavin. Intragenic complementation was found within classes rib6 and rib7. No linkage between mutations of different complementation classes was detected.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.     

Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85.

One of the negative regulators of the PHO system of Saccharomyces cerevisiae, PHO85, has been isolated by transformation and complementation of a pho85 strain. The complementing activity was delimited within a 1258 bp DNA segment and this region has been sequenced. The largest open reading frame found in this region can encode a protein of 302 amino acid residues. A pho85 mutant resulted from disruption of the chromosomal counterpart of the open reading frame described above. Therefore, we concluded that the gene we have cloned is PHO85. This result also indicates that PHO85 is nonessential. Northern analysis revealed that the size of the PHO85 message is 1.1 kb. No similarity was found between the putative amino acid sequences of two negative regulators, the PHO80 and PHO85 proteins.     

Mutations causing rapid development of Dictyostelium discoideum

A mutation affecting the speed of slime mold development has been genetically analyzed. Strain FR17 carries a recessive mutation on linkage group IV. A selection procedure for isolating more mutants of this type has been developed and new mutations have been tested for complementation. The aberrant morphology of these strains can be partially corrected by development in the presence of glucose.     

Two yeast acid phosphatase structural genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences.

We have cloned the structural genes for a regulated ( PHO5 ) and a constitutive ( PHO3 ) acid phosphatase from yeast by transformation and complementation of a yeast pho3 , pho5 double mutant. Both genes are located on a 5.1-kb BamHI fragment. The cloned genes were identified on the basis of genetic evidence and by hybrid selection of mRNA coupled with in vitro translation and immunoprecipitation. Subcloning of partial Sau3A digests and functional in vivo analysis by transformation together with DNA sequence analysis showed that the two genes are oriented in the order (5') PHO5 , PHO3 (3'). While the nucleotide sequences of the two coding regions are quite similar, the putative promoter regions show a lower degree of sequence homology. Partly divergent promoter sequences may explain the different regulation of the two genes.     

Use of Nystatin-Resistant Mutations in Parasexual Genetic Analysis in DICTYOSTELIUM DISCOIDEUM

Nystatin-resistant mutations exhibit extreme sensitivity to 1.3 mm coumarin. The mutations fall into three complementation groups so it is possible to select for nonallelic mutations conferring sensitivity to coumarin by selection on nystatin-containing nutrient agar plates. Complementation between such coumarin-sensitive mutations allows the selection of diploids on coumarin-containing nutrient agar. Two of the nystatin resistance genes, nysB and nysC, have been mapped tentatively to the previously unmarked linkage group V.     

Complementation studies withinmet-3 and the location ofmet-4 inSchizosaccharomyces pombe

The response to possible precursors of methionine was determined in 137 methionine-requiring mutants ofSchizosaccharomyces pombe. Probably, homocysteine is directly synthesized from homoserine and sulphate. One mutant, growing on synthetic medium with vitamin B12 only, indicates a side pathway. Six complementation groups were observed in a sample of 27 homocysteine-requiring mutants.Met-3 andmet-4 were found to be allelic with a distance smaller than 0.01 recombination unit, which demonstrates that the second linkage group inS. pombe, proposed earlier, is a continuation of the linkage group presented byLeupold (1958).     

人Elongator复合物Elp3亚基基因可显著补偿酵母elp3缺失菌株的生长缺陷

从HeLa细胞中分离的人的Elongator复合物在组成及与RNAPⅡ的作用方式上与酵母的Elongator复合物十分相似．但对其功能研究极少。为了研究人的Elongator复合物催化亚基Elp3的功能,将人elp3等基因转入酵母elp3基因缺失的突变菌株（elp3△菌株）,并对转化菌株进行功能互补实验和ssa和pho5基因表达分析,结果表明人elp3基因可显著恢复突变菌株对高温和Caffeine的敏感性．在低磷条件下显著补偿了突变株ph05基因表达延迟的缺陷．并可在热激条件下提高ssa3基因的表达。含酵母elp3非HAT区和人elp3 HAT区的融合yhelp3对上述缺陷有着更强的补偿能力。而HAT区催化结构域缺失的yhelp3HAT-没有任何补偿能力．表明人Elp3亚基可能与酵母的该亚基功能相似．人Elp3的HAT活性也为其行使功能所必需。     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Radiation-induced DNA strand breaks in deoxygenated aqueous solutions. The formation of altered sugars as end groups.

Gamma irradiation of DNA in deoxygenated, N2O-saturated aqueous solution leads to three bound altered sugars present as end groups in broken DNA strands. These sugars are linked to the DNA by phosphoric acid ester bonds. Two of the end groups have the structures (4) and (5). (Formula: see text) The third end group after dephosphorylation has structure (3). The formation of the bound sugars (4) and (5) is explained by a mechanism postulated earlier for the formation of free altered sugars. Except for the phosphoric acid ester linkage, the free altered sugars have the same chemical structures as the bound altered sugars.     

Fanconi anaemia in Italy: High prevalence of complementation group A in two geographic clusters

Cell fusion studies using lymphoblastoid cell lines from Fanconi anaemia (FA) patients have identified five complementation groups (FA-A to FA-E) among European FA patients. In Italy, of the 45 FA families referred to the Italian Registry of Fanconi Anaemia (RIAF), 15 took part in a project for the identification of complementation groups. Since three immortalized lymphoblast lines were resistant to a cross-linking agent, we analysed only 12 patients by complementation analysis and found that 11 belong to complementation group A. Four and seven families came from two geographic clusters in the Veneto and Campania regions, respectively, which are thought to consist of aggregates of related families in reproductive isolation. The clinical characteristics of the patients showed both intra-and interfamilial heterogeneity, although overall the disease had a relatively mild course. Since the populations in both Veneto and Campania are likely to represent genetic isolates, our finding predicts linkage disequilibrium for markers flanking theFAA gene. DNAs from these FA families may thus be utilized for positional cloning of this gene through haplotype disequilibrium mapping.     

Restoration of radiation resistance in ataxia telangiectasia cells by the introduction of normal human chromosome 11

In order to identify the human chromosome which carries a mutated gene in cells from a patient with the hereditary disorder ataxia telangiectasia belonging to complementation group D (AT-D), we performed chromosome transfer experiments via microcell fusion. A single, pSV2neo-tagged chromosome, either 11 or 12, derived from normal human fibroblasts was introduced into AT-D cells by microcell fusion, and clones which were resistant to the antibiotic G418 were isolated. All 3 hybrid clones containing an additional copy number of chromosome 11 showed a restoration of the resistance of wild-type cells to killing by X-irradiation, whereas all 3 hybrid clones containing an additional copy number of chromosome 12 remained hyper-radiosensitive, like the parental AT cells. The results indicate that a defective gene of AT-D cells is also located on chromosome 11, since a genetic linkage analysis has previously suggested that a defective gene of its complementation group A is located on this chromosome.