Effects of pyrimidine enriched medium on the female sterile mutants,Rudimentary andSonless inDrosophila

The effect of pyrimidine-enriched medium on the expression of the sex-linked female sterile mutationsrudimentary (r) andsonless (snl) ofDrosophila melanogaster was investigated. Although pyrimidine supplementation did significantly improve the fertility ofr females, as others have previously reported, it had no effect onsnl females. The total ribonucleic acid (RNA) content of eggs produced byr, snl, and control females was also measured. Eggs produced from homozygousr females mated tor males contained less total RNA than did eggs from other matings. These results support the hypothesis that certain sex-linked female sterile mutations cause the production of eggs deficient in some respect. In the case ofr, the deficiency seems to by pyrimidines. The biochemical deficiency associated withsnl requires further investigation.     

Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site

Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities. Most of the mutants show good transport of melibiose but have lost the recognition of TMG. In addition, most mutants show little or no transport of lactose. Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose. The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid. The mutations were located on helices I, IV, VII, X and XI. We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel.     

Mutagenic potency and specificity of procarbazine in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa.

Procarbazine (Natulan) was tested for its mutagenic potency and specificity in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In these experiments, procarbazine was a weak mutagen when present in growing cultures but nonmutagenic when conidial suspensions (nongrowing conidia) were treated. A total of 208 ad-3 mutants recovered after exposure of growing cultures of H-12 to 1 mg of procarbazine/ml, and 2 ad-3 mutants of spontaneous origin, were characterized genetically. These tests distinguish among gene/point mutations (ad-3R) at the ad-3A or ad-3B locus, multilocus deletion mutations ([ad-3]IR) covering one or more loci in the ad-3 and immediately adjacent regions, and 3 different classes of multiple-locus mutations: gene/point ad-3 mutations with a recessive lethal mutation elsewhere in the genome (ad-3R + RL), gene/point mutations with a closely linked recessive lethal mutation (ad-3R + RLCL), and multilocus deletion mutations with a closely linked recessive lethal mutation ([ad-3]IR + RLCL). All of the procarbazine-induced ad-3 mutants resulted from gene/point mutations; 92.2% (200/217) resulted from gene/point mutations at the ad-3A or ad-3B locus, and 3.7% (8/217) resulted from gene/point mutations with a recessive lethal mutation elsewhere in the genome. Identical percentages (15.4% [20/130] and 15.4% [12/78]) of the sigma ad-3BR and sigma ad-3AR mutants were leaky, and a high percentage (71.5% [93/130]) of the sigma ad-3BR mutants had nonpolarized complementation patterns. These results indicate that procarbazine-induced ad-3 mutants of Neurospora crassa are composed solely of gene/point mutations (ad-3R) that resulted, predominantly or exclusively, from base-pair substitutions. The Neurospora specific-locus data on procarbazine-induced ad-3 mutants are compared with data from similar experiments with the mouse using the morphological specific-locus assay; marked similarities were found between the mutagenic effects of procarbazine in the 2 specific-locus assays.     

Meiosis in NEUROSPORA CRASSA. I. the Isolation of Recessive Mutants Defective in the Production of Viable Ascospores

A scheme has been devised for efficient isolation of recessive meiotic mutants of Neurospora crassa. These mutants were detected by their reduced fertility or by the abortion of ascospores. Their isolation involved the selection and screening of the strains arising from ascospores disomic (n + 1) for linkage group I (LG I), which bears the mating-type locus. These strains are self-fertile heterokaryons that contain two types of haploid nuclei of opposite mating types (A + a). Selfings of these strains are homozygous for genes on all linkage groups except LGI and therefore allow the expression of recessive mutants with an altered sexual cycle. Using this selection procedure, three classes of mutants were detected. In one class, mutants had an early block in perithecial development (class I), and in another mutants had altered perithecia, but apparently unaltered fertility (class III). No recessive mutants were observed and all mutants tested (eight of class I and two of class III) were expressed only when used as the maternal parent. A third mutant class displayed normal production of perithecia, but defective formation of asci (class IIA), or black ascospores (class IIB). Four of 13 class IIA mutants were analyzed, and two of them [asc(DL131) and asc (DL400)] were definitely recessive analysis of 10 of 13 class IIB mutants disclosed six recessive, mutually complementing mutants: ase(DL95), asc(DL243), asc(DL711), asc(DL879), asc(DL917m) and asc(DL961). Mutants asc(DL95), asc(DL243) and the previously studied mei-1 mutant (Smith 1975) complemented one another in crosses, but did not recombine. These may be alleles of the same gene, or they may comprise a gene cluster.     

Identification and Gene Mapping of a Novel Mutant supernumerary lodicules(snl) in Rice

In order to gain a better understanding of rice flower development, a rice flower mutant supernumerary lodicules (snl), which was identified from ethyl methane sulfonate (EMS)-treated Jinhui10 (Oryza sativa L. ssp. indica) was used in the present study. In the snl mutant, the palea obtained lemma identity, additional glume-like organs formed, lodicules increased and elongated, stamens decreased, and a few aberrant carpels formed. These phenotypes suggest that SNL is involved in the entire rice flower develo...     

Mutations partially inactivating the lactose repressor of Escherichia coli

After treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 133 independent mutants of a haploid strain of Escherichia coli able to use phenyl-beta-galactoside as a carbon source were obtained. The galactoside was specific in selecting for mutants with increases in their uninduced levels of beta-galactosidase. Virtually all mutants (37 in a subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) repressed beta-galactosidase synthesis 4 to 50 times less than wild type, but were more numerous in the lower part of this range. Their beta-galactosidase was inducible to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine growth conditions. The decreased activity did not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors appeared to carry UAG or UGA chain-terminating codons. On the assumption that the partially defective repressor mutants carried missense mutations, it is argued that missense mutations in the lac repressor gene modify the repressor's affinity for the operator with high probability. An explanation is proposed for the apparent sensitivity of this repressor function to partial inactivation as the result of amino acid substitutions.     

Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.     

Characterization of strong polar mutations in a region immediately adjacent to the L-arabinose operator in Escherichia coli B-r

Seven l-arabinose-negative mutations are described that map in three genetically distinct regions immediately adjacent to the araO (operator) region of the l-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dominant, trans-recessive polarity effect upon the expression of l-arabinose isomerase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initiator region of the l-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens diethyl sulfate and ICR-191. Three of these mutants are suppressible by two independently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara(+) revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppressors, and their partially polar revertants are discussed in terms of the mechanism of initiation of expression of the l-arabinose operon.     

Molecular and Genetic Analyses of the B Type Surface Protein Gene from Paramecium Tetraurelia

The gene encoding the B type variable surface protein from Paramecium tetraurelia stock 51 has been cloned and sequenced. The 7,182 nucleotide open reading frame contains no introns and encodes a cysteine-rich protein that has a periodic structure including three nearly perfect tandem repeats in the central region. Interestingly, the B gene is located near a macronuclear telomere as was shown previously for two other paramecium surface protein genes. In this paper, we characterize four independent mutants with complete macronuclear deletions of the B gene. Previous analysis of different macronuclear deletion mutants of the A surface protein gene demonstrated two types of inheritance: typical Mendelian segregation (as illustrated by d12) and cytoplasmic inheritance (shown by d48). F(1) analysis of four B(-) mutants crossed with wild-type cells reveals heterozygous F(1) cell lines derived from both parental cytoplasms contain approximately the same copy number of the B gene, as expected for a recessive Mendelian mutation. Analysis of F(2) progeny from three of these four B(-) mutant crosses indicates that one of the three exhibits a Mendelian 1:1 segregation ratio of B(+) and B(-) cell lines. The other two show a preponderance of B(+) cells, but this is not correlated with the parental cytoplasmic type. In addition to having a large number of B(+) individuals, the d12.144, A(-), B(-) mutant produced some F(2) progeny that stably maintain less than normal macronuclear amounts of the A gene and/or the B gene.     

The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.     

Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

Arabidopsis Paired Amphipathic Helix Proteins SNL1 and SNL2 Redundantly Regulate Primary Seed Dormancy via Abscisic Acid–Ethylene Antagonism Mediated by Histone Deacetylation

Histone (de)acetylation is a highly conserved chromatin modification that is vital for development and growth. In this study, we identified a role in seed dormancy for two members of the histone deacetylation complex in Arabidopsis thaliana, SIN3-LIKE1 (SNL1) and SNL2. The double mutant snl1 snl2 shows reduced dormancy and hypersensitivity to the histone deacetylase inhibitors trichostatin A and diallyl disulfide compared with the wild type. SNL1 interacts with HISTONE DEACETYLASE19 in vitro and in planta, and loss-of-function mutants of SNL1 and SNL2 show increased acetylation levels of histone 3 lysine 9/18 (H3K9/18) and H3K14. Moreover, SNL1 and SNL2 regulate key genes involved in the ethylene and abscisic acid (ABA) pathways by decreasing their histone acetylation levels. Taken together, we showed that SNL1 and SNL2 regulate seed dormancy by mediating the ABA-ethylene antagonism in Arabidopsis. SNL1 and SNL2 could represent a cross-link point of the ABA and ethylene pathways in the regulation of seed dormancy.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.