An unstable nuclear gene in phycomyces

A gentic instability in Phycomyces is described that appears to be associated with a single nuclear gene, dar. The wild type is able to take up riboflavin and its toxic analogue, deaza-riboflavin, from nanomolar concentrations in the medium. The mutants are unable to take up riboflavin and are resistant to deaza-riboflavin. Forward and reverse mutation rates are estimated to be 4 x 10^-5 and 2 x 10^-3 per nuclear division. Independently arisen dar mutants do not complement in heterokaryons. The mutant alleles are almost completely recessive. The phenotype of spores is not determined cell-autonomously, but is strongly influenced by the allele ratio among the nuclei in the sporangium of origin.     

A Comparison of Mutation Rates for Specific Loci and Chromosome Regions in Dysgenic Hybrid Males of DROSOPHILA MELANOGASTER

The mutation rates of specific loci and chromosome regions were estimated for two types of dysgenic hybrid males. These came from crosses between P or Q males and M females in the P-M system of hybrid dysgenesis. The M x P hybrids were the more mutable for each of the loci and chromosome regions tested. The Beadex locus was highly mutable in these hybrids but did not mutate at all in the sample of gametes from the M x Q hybrids. The singed locus had 75% of the mutability of Beadex in the M x P hybrids; it was also mutable in the M x Q hybrids. The white locus was only slightly mutable in the M x P hybrids and not at all mutable in the M x Q hybrids. The mutations in singed and white probably arose from the insertion of P elements into these loci; the mutations at Beadex probably involved the action of a P element located near this locus on the X chromosome of the P strain that was used in the experiments. Mutations in two chromosome regions, one including the zeste-white loci and the other near the miniature locus, were much more frequent in the M x P hybrids than in the M x Q hybrids. These mutations also probably arose from P element insertions. The implication is that insertion mutations occur infrequently in the M x Q hybrids, possibly because most of the P elements they carry are defective. In M x P hybrids, there is variation among loci with respect to P elements mutagenesis, indicating that P elements possess a degree of insertional specificity.     

Selective Recombination System in Bombyx mori. I. Chromosome Specificity of the Modification Effect

The effect of modifiers on recombination frequency between Ze and lem loci on chromosome 3 to elucidate the chromosome specificity of modification and the distribution of modifiers using Bombyx mori lines selected for high (H) and low (L) recombination rates between the p^S and Y loci in chromosome 2 was investigated. By crossing to the Z (Ze lem/++) line, the recombination rate between the p^S and Y loci in chromosome 2 was decreased from 28.18 to 23.33 in the H line and was increased from 4.92 to 16.05 in the L line. On the other hand, the recombination rate between the Ze and lem loci in chromosome 3 was increased from 16.21 to 20.21 in the Z line by crossing to the H line, but also increased to 19.02 by crossing to the L line. The significant correlation observed between the transformed recombination rates of chromosomes 2 and 3 in the (Z x L) x L backcross indicated that there were common factors modifying recombination frequency in chromosomes 2 and 3 or different factors linked to the same chromosomes. In the family of L x [(Z x L) x L] backcross, the distribution of transformed recombination rates indicated that there were several factors in the remaining chromosomes which were modifying recombination frequency in chromosome 2 but not in chromosome 3. It was also indicated that these factors were linked to different chromosomes than are the factors modifying recombination frequency in chromosome 3. In order to interpret these results, one genetic system model controlling recombination that consists of general and local recombination modifiers was proposed. The evolution of dynamic genetic systems that would effectively reduce recombinational load without reducing the advantage of recombination was discussed.     

The genetic system controlling recombination in the silkworm

The nature of recombination modifiers was investigated in Bombyx mori lines selected for high (H) and low (L) recombination rates between the p^S and Y loci in chromosome 2. Since the mean recombination rates for the H x L and L x H F₁ crosses were approximately intermediate between those of high and low lines, the cytoplasmic maternal effect and difference in the activity of recombination modifiers between marked and unmarked second chromosomes were not detected. The H x (L x H), H x (H x L), L x (L x H) and L x (H x L) backcrosses indicated the presence of additive and dominance effects of marked and unmarked second chromosomes and the remaining chromosomes.——Recombination rates between the p^S and Y loci in chromosome 2 and half-nonrecombination rates between the pe and re loci in chromosome 5 of high and low lines indicated that these recombination modifiers caused changes in the recombination frequency between p^S and Y in chromosome 2, but not between pe and re in chromosome 5.——There were no differences in viability between individuals having the second chromosomes of the recombinant types [p^S +, p^Y (H); p^S +, + Y (L)] and those of the nonrecombinant types [p^S Y, p + (H); p^S Y, + + (L)] in both high and low lines. Mean recombination rates measured in cis [p^S Y/p + (H); p^S Y/+ + (L)] and trans [p^S +/p Y (H); p^S +/+ Y (L)] males were the same in the high but not in the low line. No segregation of a single recombination modifier was indicated by the distribution of recombination rates measured in trans males [p^S +/p Y (H); p^S +/+ Y (L)] of high and low lines. Accordingly, the recombination modifiers distributed on chromosome 2 in the heterozygous condition were not gross chromosomal aberrations, but polygenic factors in the low line.     

Genetic Effects of Acute Spermatogonial X-Irradiation of the Laboratory Rat

The genetic effects of one generation of spermatogonial X-irradiation in rats, by a single dose of 600r in one experiment and by a fractionated dose of 450r in another, were measured in three generations of their descendants. Estimates of dominant lethal mutation rates—(2 to 3) x 10 ^-4/gamete/r—from litter size differences between irradiated and nonirradiated stock were consistent with previous estimates from rats and mice. Similar consistency was found for estimates of sex-linked recessive mutation rates—(1 to 2) x 10^-4 chromosome/r—from male proportions within strains; however, when measured in crossbreds the proportion of males was higher in the irradiated than in the nonirradiated lines. This inconsistency in results is in keeping with the contradictory results reported for recessive sex-linked lethal mutation rates in mice. The effects used to estimate recessive lethal mutation rates which were unusually high—(2 to 14) x 10^-4/gamete/r—were not significant. Other factors that could have contributed to the observed effects are postulated.     

Genetic Mechanisms of Rare Matings of the Yeast SACCHAROMYCES CEREVISIAE Heterozygous for Mating Type

Yeast heterozygous for mating type lacks the ability to conjugate as judged by the mass-mating technique and accordingly is designated "non-mater". However, the non-mater shows rare mating ability with a frequency of less than 10^-6. In the present study, the RD auxotroph mating method was mainly employed with the intention of examining the rare mating ability of various non-maters, using lactate ethanol minimal medium as a selective medium for hybridization. Crosses of aα x a, aα x a, aaα x a, aαα x a, etc. resulted in the production of respective hybrids with a relatively high frequency of about 10^-6 to 10^-7, whereas crosses of aaα x a, aαα x α, aaαα x a, aaαα x α, etc. resulted in hybrids with an extremely low frequency of about less than 10^-8. Genetic analyses revealed that the rare matings were mostly caused by the presence of cells derived from the non-maters in which mating type had converted to a homozygous genotype. Mitotic recombination was shown to be a likely explanation for most of the conversion, judging from associated exchange of an outside marker, thr4. By successive employment of the RD auxotroph mating method, it was possible to produce a series of polyploid yeasts, triploids to octoploids. The DNA content and the cell volume were observed to increase parallel to the elevated ploidy states.     

Chloroplast and Mitochondrial DNA Variation in HORDEUM VULGARE and HORDEUM SPONTANEUM

A survey of restriction fragment polymorphism in Hordeum vulgare and Hordeum spontaneum was made using 17 and 16 hexanucleotide restriction endonucleases on chloroplast (cp) and mitochondrial (mt) DNA, respectively. The plant accessions originated from various places throughout the Fertile Cresent and Mediterranean. The types of changes in cpDNA consisted of nucleotide substitutions and insertions and deletions on the order of 100 base pairs. In contrast, mtDNA has most likely undergone larger insertions and deletions of up to 20 kilobase pairs in addition to rearrangements. Grouping of mtDNA fragment data showed that in some cases geographical affinities existed between the two species, whereas in others there were no clear affinities. Nucleotide diversity estimates derived from the restriction fragment data were used in a number of comparisons of variability. Comparisons of overall mtDNA variability (nucleotide diversity = 9.68 x 10^-4) with cpDNA variability (nucleotide diversity = 6.38 x 10^-4 ) indicated that the former are somewhat more variable. Furthermore, there was no indication that the wild H. spontaneum (cpDNA diversity = 5.57 x 10^-4; mtDNA diversity = 6.04 x 10 ^-4) was more variable than the land races of H. vulgare (cpDNA diversity = 5.88 x 10^-4; mtDNA diversity = 9.79 x 10^-4). In fact, on the basis of mtDNA diversity, H. vulgare was the more variable species. Comparison of organelle nucleotide diversity estimates with an estimate of nuclear nucleotide diversity derived from existing isozyme data provided evidence that both organelle genomes are evolving at a slower rate than the nuclear genome.     

Quantitative Measurement of the Ability of Different Mutagens to Induce an Inherited Change in Phenotype to Allow Maltose Utilization in Suspension Cultures of the Soybean, GLYCINE MAX (L.) Merr

Using a newly developed plating system, we have measured cell survival and the frequencies of variation in an inherited trait after treatment of soybean cell suspensions with different mutagens: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG), hycanthone (1-{[2-(diethylamino) ethyl] amino}-4-(hydroxymethyl)-9H-thioxanthen-9-one and ultraviolet light (UV).—The heritable variation selected for displays a phenotype of rapid growth on maltose as carbon source. The marker is stable in the absence of maltose, and prolonged growth of variant cells on sucrose has not shown reversions to slow growth. Doubling time in suspension cultures is decreased from 100 hr to ca. 30 hr by the mutation. Both wild-type and variant cells grow on sucrose with a 24-hr doubling time. Thus, lethality after mutagen treatment can be estimated rapidly by growth on sucrose, whereas variants are scored on maltose medium. The spontaneous frequency of variants was 1.2 x 10^-7; induced frequencies ranged from a low of 3.6 x 10^-5 for EMS to a high of 10^-3 for hycanthone. The high frequency of variants induced by hycanthone, a frame-shift mutagen, and the observation that UV induces variants in haploid cells with much higher frequency than in diploid cells suggests a recessive mutation.     

Intraspecific Variation of Ribosomal Gene Redundancy in ZEA MAYS

Ribosomal genes in eukaryotes are highly redundant. Considerable variation in the level of redundancy among species, especially in higher plants, has been reported; but except for deletion and duplication mutants, it is generally accepted that intraspecific variability in redundancy level is small. We have examined the level of redundancy in several lines of maize by DNA-rRNA saturation hybridization. The amount of nuclear DNA which hybridizes with rRNA in the ten lines examined varied from 0.24% to 0.50%. The number of rRNA genes per diploid genome thus ranges from 1.12 x 10⁴ to 2.32 x 10⁴. Results also indicate that the level of redundancy is genetically transmitted.     

Distribution and Frequency of Tandem Duplications of the rII Region of Bacteriophage T4d

Genetic analyses of 49 duplications of the rII region of bacteriophage T4D suggests that there is a non-random relationship between the end points of duplicated segments, that relaxed packaging restrictions have little if any effect on the distribution of duplications, that segregation is 3–4 times more frequent than normal recombination for the same interval, and that non-tandem duplications are rare. Extrapolation of the r1231 x rJ101 cross data suggests that the minimum frequency of duplications/genome is 1.7 x 10^-6, but possibly 3.4 x 10^-4.     

Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia

Despite its morphological similarity to the other species in the Drosophila melanogaster species complex, D. sechellia has evolved distinct physiological and behavioral adaptations to its host plant Morinda citrifolia, commonly known as Tahitian Noni. The odor of the ripe fruit of M. citrifolia originates from hexanoic and octanoic acid. D. sechellia is attracted to these two fatty acids, whereas the other species in the complex are repelled. Here, using interspecies hybrids between D. melanogaster deficiency mutants and D. sechellia, we showed that the Odorant-binding protein 57e (Obp57e) gene is involved in the behavioral difference between the species. D. melanogaster knock-out flies for Obp57e and Obp57d showed altered behavioral responses to hexanoic acid and octanoic acid. Furthermore, the introduction of Obp57d and Obp57e from D. simulans and D. sechellia shifted the oviposition site preference of D. melanogaster Obp57d/e^KO flies to that of the original species, confirming the contribution of these genes to D. sechellia's specialization to M. citrifolia. Our finding of the genes involved in host-plant determination may lead to further understanding of mechanisms underlying taste perception, evolution of plant–herbivore interactions, and speciation.     

Spontaneous Unstable UNC-22 IV Mutations in C. ELEGANS Var. Bergerac

This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 x 10^-4 , and most of these spontaneous unc-22 mutations revert at frequencies between 2 x 10^-3 and 2 x 10 ^-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10^-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable.

The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

     

Killer Phenomenon in USTILAGO MAYDIS: The Organization of the Viral Genome

The double-stranded RNA content, the production of inactive killer protein, and the presence of virus-like particles were examined in induced nonkiller mutants and nonkiller progeny from a cross between a killer strain and a sensitive strain. A correlation between the loss of the 0.7 x 10⁶ daltons dsRNA of the Ustilago maydis P6 virus and the lack of synthesis of the killer protein was established. In vitro and in vivo complementation between nonkiller strains provide additional support for the suggestion that the 0.7 x 10⁶ daltons dsRNA is related to the killer function. The coding capacity of the various species of dsRNA is discussed in relation to their possible function.     

The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.     

Genetic Map of the Lactose Repressor Gene (i) of ESCHERICHIA COLI

39 tonB i^- deletions were used to map 57 independently isolated i gene mutants. Methods selective for i⁺ recombinants from various types of i mutants are described. i⁺ recombination frequencies of 6 x 10^-6 to 6 x 10^-7 can be detected in the different selective systems. Twenty i^s mutations and 12 i^-d mutations map in distinct but different regions of the gene. i^-_sus mutations are scattered over the gene.     

High Mutability in Male Hybrids of DROSOPHILA MELANOGASTER

The frequencies of sex-linked lethal mutations arising in hybrid male offspring from various crosses and in nonhybrid controls were determined. The hybrids were produced by crossing representative strains of the P-M system of hybrid dysgenesis in all possible combinations. Males from the cross of P males x M females had a mutation rate about 15 times higher than that of nonhybrid males from the P strain. Genetically identical males from the reciprocal cross had a mutation rate 3 to 4 times that of the nonhybrids. For crosses involving a Q strain, a significant increase in the mutation rate was detected in males produced by matings of Q males with M females. No increase was observed in genetically identical males from the reciprocal mating. Crosses between P and Q strains gave male hybrids with mutation rates not different from those of nonhybrids. Many of the lethals that occurred in hybrids from the cross of P males x M females appeared to be unstable; fewer lethals that arose in hybrids from the cross of Q males x M females were unstable. The relationship between P and Q strains is discussed with respect to a model of mutation induction in dysgenic hybrids.     

Frequency of gamma-Ray Induced Specific Locus and Recessive Lethal Mutations in Mature Germ Cells of the Zebrafish, BRACHYDANIO RERIO

Specific locus and recessive lethal mutations are induced by γ-rays with approximately first order kinetics in the zebrafish (Brachydanio rerio) with frequencies of 4 x 10^-5 r^-1 and 4 x 10^-3 r^-1, respectively. The surprisingly low ratio (100:1) of recessive lethals to specific locus mutations may be due to the induction of large deficiencies by γ-rays.     

Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli

Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.     

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.