Site-Specific Instability in DROSOPHILA MELANOGASTER: The Origin of the Mutation and Cytogenetic Evidence for Site Specificity

During a study of delayed mutations, an unstable X chromosome (Uc) was detected. Spontaneous X-linked recessive lethal mutations were detected in 34 of 993 sperm sampled from 50 males carrying this chromosome. All but three of the 34 lethals originated as clusters in three of the 50 males Cytogenetic and complementation analyses revealed 14 intrachromosomal rearrangements: ten inversions, two reverse repeats, one deficiency and one transposition. Eight of the 14 rearrangements have one break in the 6F1-2 doublet and two rearrangements have a break in 6F1-5 of the X chromosome. The remaining four rearrangements have in addition to the aberrations a lethal point mutation between 6F1 and 6F5. Though each of the lethal lines was established from a single lethal-bearing female, chromosome polymorphism is evident in 17 of the 18 lines having rearrangements, with certain aberrations recurring in several lines. The lethal mutations revert frequently to the nonlethal state, and cytological evidence indicates that more than one mutational event may occur at the unstable locus of the chromosome during one generation. Two lethal lines had more than one type of chromosome rearrangement sharing a common breakpoint. These observations are consistent with the view that the instability of the Uc lines is caused by a transposable element capable of site-specific chromosome breaks and perpetual generation of mutations. The mutagenic and genetic properties of transposable elements can be related to the two-mutation theory of KNUDSON (1971) for cancer initiation.     

Cytogenetics of Notch Mutations Arising in the Unstable X Chromosome Uc of Drosophila melanogaster

A derivative of the unstable X chromosome, Uc, isolated in 1978 is still unstable and exhibits most of the genetic properties characteristic of the original Uc. This derivative, Df(1)cm-In, contains an inversion of the genes between bands 6F1-2 and 3D3-5 and a lethal deficiency between 6D5-7 and 6F1-2. This chromosome generated Notch mutations at a rate of 3.47 +/- 0.32% during seven consecutive generations. Cytological analysis of 50 Notch mutations of independent origin in the Df(1)cm-In chromosome showed that all of the 50 had an apparently identical deletion involving the region between 3D3-5 and 3C7-8 of the X chromosome. The results of in situ hybridization indicated that the extent of deletion in all of the 20 Notch deficiencies sampled from the 50 mentioned above involves about 10 kb of the sequences from the 3' end of the Notch locus. In addition to hypermutability and the accumulation of site-specific chromosome breaks, the Df(1)cm-In chromosome reinverts its inversion to the normal sequence and exhibits use of the existing chromosome breakpoints to generate new rearrangements.     

Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.     

Analysis of Dysgenesis-Induced Lethal Mutations on the X Chromosome of a Q Strain of DROSOPHILA MELANOGASTER

The Q strain known as v6 was tested for its ability to induce X-linked lethal mutations in male and female hybrids from crosses with M strains in the P-M system of hybrid dysgenesis. All measurements of the mutation rate were made on the X chromosome derived from the v6 strain. The lethal rate for young hybrid males from the cross M female X v6 male was 1.11% per chromosome. For older males, it was only 0.44%, suggesting that there is less mutational or more repair activity in the germ cells of the older males or that mutant cells are selectively eliminated as the hybrid males age. The lethal rate for hybrid females from comparable crosses was approximately the same for both ages that were tested. However, it was substantially less than the rate for the hybrid males--only 0.26% per chromosome. Genetically identical hybrid females from reciprocal crosses also showed a low mutation rate, 0.13% per chromosome. Again, there was no difference between young and old flies. Mapping experiments established that most of the lethal mutations that were recovered from the male and female hybrids were located in two regions on the X chromosome, one between bands 14B13 and 15A9 , the other between bands 19A1 and 20A , which encompasses the maroonlike locus. More refined mapping of the lethals in the maroonlike region demonstrated that the vast majority of these affected a single gene located in band 19C4 . Cytological analysis of the lethal chromosomes revealed that several carried rearrangements, including inversions, duplications and deficiencies. Chromosome breakage occurred primarily in bands 14D1 -3 and 18F- 20A , and most of the breaks in the latter segment were located in 19C . However, rearrangements involving 19C and mutations of the gene in 19C4 were mutually exclusive events. In situ hybridization of a P element probe to the chromosomes of v6 demonstrated that P elements reside at a minimum of five sites on the X chromosome. These P element sites correspond to the mutational and breakage hot spots on that chromosome. The combined genetic and cytological data imply that most of the X-linked lethal mutations that occur in M X v6 hybrids are due to local P element action. Consideration of these and other data suggest that v6 is a weak P strain in the P-M system of hybrid dysgenesis and that other Q strains might also be regarded in this way.     

Conditional lethal mutations shift the genome from stability to instability

The phenomenology of genomic destabilization is described in Drosophila melanogaster mutants containing radiation-induced conditional dominant lethals in the X chromosome and in autosome 2. Destabilization manifests itself as (1) the loss or decrease of lethality of previously lethal mutations; (2) the loss of expression of visible dominant mutations in an opposite homolog; (3) chromosomal instability resulting in the loss of the X chromosome in germline and somatic cells; (4) the occurrence of novel mutations (secondary mutagenesis); (5) the occurrence of single and mass modifications; (6) disturbances in individual development (formation of morphoses). The key event for the shift of the genome from the stable state into the unstable one is the occurrence of a conditional dominant lethal mutation.     

Chromosomal superkiller mutants of Saccharomyces cerevisiae.

Yeast strains carrying a 1.5 X 10(6)-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e., increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated ski1 through ski4 (for superkiller), ski3 and ski4 are located on chromosome XIV, and ski1 is on chromosome VII. A ski1 mutation results in a decreased rate of cell growth. The kex1 and kex2 mutations are epistatic to each ski mutation.     

Hybridization studies on Blattella germanica and B. asahinai (Dictyoptera: Blattellidae): species divergence and a possible influence of a major chromosome mutation.

An earlier study indicated that Blattella asahinai is separated from its close relative B. germanica by a non-reciprocal translocation that apparently involved the transfer of the nucleolus organizing region from the X chromosome of B. germanica or a B. germanica like ancestor to chromosome 12 in B. asahinai. Continued study on divergence of the two species included genetic analyses of fecundity, egg case hatch, nymphal hatch, sex ratios, and segregation of X chromosomes and the segment carrying the B. asahinai nucleolar organizing region in interspecific and backcross matings. Overall, a complex of maternally related disadvantages was associated with B. asahinai. The effects of cytoplasmic factors could not generally be distinguished from possible effects of X chromosome - cytoplasmic interactions. In two crossing systems, the data fit a hypothesis of lethal effects from the presence of an X chromosome in alien cytoplasm. Cytologic differences occurred frequently in backcrosses, especially with F1 hybrid females, but were limited to chromosomes and chromosome segments affected by the translocation. The possible relationship of the chromosome mutation to traits affecting reproduction and its role in species divergence are discussed.     

Trenimon-induced sex chromosome loss in Drosophila: different dose-responses for ring-X loss as compared to rod-X loss and Y-marker loss

Drosophila melanogaster males carrying either a ring- or a rod-shaped X-chromosome were injected or fed with Trenimon (triaziquone) at concentrations ranging from 5 X 10(-5) to 2 X 10(-2) mM. The F1 generation was assayed for the occurrence of total sex chromosome loss and of Y-chromosome markers. Sex-linked recessive lethal tests were carried out simultaneously. The data show that significant induction of ring-X loss occurs already at very low treatment concentrations (5 X 10(-5) -10(-4) mM) whereas rod-X loss or Y-marker loss is only seen at 2-5 X 10(-3) mM and higher. Induction of sex-linked recessive lethals is observed from 10(-4) -10(-3) mM on. These results add to existing evidence that loss of ring-X chromosomes, induced by some chemicals, may proceed by a mechanism different from the kind of events leading to chromosome breakage, as measured by rod-X loss and Y-marker loss.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. I. Origin and Molecular Mapping of Radiation- and Chemical-Induced Lethal Brown Deletions

Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.     

The distribution of randomly recovered X-ray-induced sex-linked genetic effects in Drosophila melanogaster

Cytogenetic analysis of more than 1500 randomly recovered lethal X chromosomes derived from 2000 and 3000 r X-ray exposures of post-meiotic male germ cells has made possible a plot of the distribution in different regions of the X chromosome of: (1) gene mutations associated with cytologically normal chromosomes, (2) mutations associated with chromosomal rearrangement breakpoints, (3) deficiencies, and (4) rearrangement breakpoints whether or not they are associated with mutations. The distribution of point mutations, vital loci and rearrangement breakpoints in different regions of the X chromosome is not proportional to either the number of bands or the relative DNA content. Further, the density of vital loci (those capable of mutating to a lethal allele) is quite different in some regions as compared to others. For example, vital loci in the 3AB region, which has been thoroughly studied by Judd and others, are at least as numerous as bands; whereas, the 3CD region, equally long, has only two vital loci. Other regions densely populated with vital loci include 1B, 1F-2A, 10A, 11A, and 19EF; sparsely populated regions include 6EF and 10B-10E. It seems reasonable to conclude that the recovered X-ray-induced mutants available for analysis do not represent a random sample of those initially induced in the exposed male germ cells.     

Genetic Instability in Drosophila Melanogaster Mediated by Hobo Transposable Elements

Eight independent recessive lethal mutations that occurred on derivatives of an unstable X chromosome (Uc) in Drosophila melanogaster were analyzed by a combination of genetic and molecular techniques. Seven of the mutations were localized to complementation groups in polytene chromosome bands 6E; 7A. In situ hybridization and genomic Southern analysis established that hobo transposable elements were associated with all seven of the mutations. Six mutations involved deletions of DNA, some of which were large enough to be seen cytologically, and in each case, a hobo element was inserted at the junction of the deletion's breakpoints. A seventh mutation was associated with a small inversion between 6F and 7A-B and a hobo element was inserted at one of its breakpoints. One of the mutant chromosomes had an active hobo-mediated instability, manifested by the recurrent production of mutations of the carmine (cm) locus in bands 6E5-6. This instability persisted for many generations in several sublines of an inbred stock. Two levels of instability, high and basal, were distinguished. Sublines with high instability had two hobo elements in the 6E-F region and produced cm mutations by deleting the segment between the two hobos; a single hobo element remained at the junction of the deletion breakpoints. Sublines with low instability had only one hobo element in the 6E-F region, but they also produced deletion mutations of cm. Both types of sublines also acquired hobo-mediated inversions on the X chromosome. Collectively, these results suggest that interactions between hobo elements are responsible for the instability of Uc. It is proposed that interactions between widely separated elements produce gross rearrangements that restructure the chromosome and that interactions between nearby elements cause regional instabilities manifested by the recurrence of specific mutations. These regional instabilities may arise when a copy of hobo transposes a short distance, creating a pair of hobos that can interact to produce small rearrangements.     

Interlinear Differences in the Morphology of the Pericentric Region of Salivary Gland Polytene X Chromosome ofDrosophila melanogaster

Morphology of the Drosophila melanogasterpolytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ESmutation has been examined. In all of the strains carrying the Su(UR)ESmutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by -heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ESmutants the most proximal X chromosome euchromatic gene,su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20. All large bands observed in section 20 of the w; Su(UR)ESstrain were also present inIn(1)sc ⁴; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Horka, a Dominant Mutation of Drosophila, Induces Nondisjunction And, through Paternal Effect, Chromosome Loss and Genetic Mosaics

Fs(3) Horka (Horka) was described as a dominant female-sterile mutation of Drosophila melanogaster. Genetic and cytological data show that Horka induces mostly equational nondisjunction during spermatogenesis but not chromosome loss and possesses a dominant paternal effect: the X, second, third and the fourth chromosomes, but not the Y, are rendered unstable while undergoing spermatogenesis and may be lost in the descending zygotes. The frequency of Horka-induced chromosome loss is usually 2-4% but varies with the genetic background and can be over 20%. The X chromosome loss occurs during the gonomeric and the initial cleavage divisions. Loss of the X and fourth chromosomes shows no correlation. We propose, based on similarities in the mutant phenotypes with the chromosome destabilizing mutations nonclaret disjunctional and paternal loss, that the normal Horka(+) product is required for function of the centromeres and/or nearby regions. Horka is a convenient tool for the generation of gynandromorphs, autosome mosaics and for the study of gene expression in mosaics.     

Meiotic studies in mice carrying the sex reversal (Sxr) factor

A sex reversal factor (Sxr) that causes mice having apparently normal X chromosomes to become phenotypically male is transmitted in an autosomal pattern. The origin of the Sxr factor is still unknown. It seems most likely that it has originated from an autosomal gene mutation or is the result of a translocation of part of the Y chromosome to one of the autosomes. Chromosomes from four XY and six XO mice carrying this sex reversal factor were examined in the diakinesis stage of meiosis. The following unusual observations were noted: (1) in XY males carrying the Sxr factor, the X and Y chromosomes were separated more often than in controls. (2) The Y chromosome tends to be closer to an autosome when the X and Y are separate than when the X and Y are attached. (3) A chromosome fragment was present in 4/226 cells from two XO males and a single cell from an XY, Sxr carrier. Although there is no direct evidence, these observations seem to favor the possibility that the Sxr factor involves a chromosomal rearrangement rather than a single gene mutation.     

Isolation of Dominant Xo-Feminizing Mutations in Caenorhabditis Elegans: New Regulatory Tra Alleles and an X Chromosome Duplication with Implications for Primary Sex Determination

A strain of Caenorhabditis elegans was constructed that permits selection of dominant or sex-linked mutations that transform XO animals (normally male) into fertile females, using a feminizing mutation, tra-2(e2046gf), which by itself does not sexually transform XO males. Twenty-three mutations were isolated after chemical mutagenesis and found to fall into both expected classes (four dominant tra-1 mutations and eight recessive xol-1 mutations) and novel classes. The novel mutations include 10 second-site mutations of tra-2, which are called eg mutations, for enhanced gain-of-function. The tra-2(gf, eg) alleles lead to complete dominant transformation of XO animals from fertile male into fertile female. Also isolated was a duplication of the left end of the X chromosome, eDp26, which has dominant XO lethal and feminizing properties, unlike all previously isolated duplications of the X chromosome. The properties of eDp26 indicate that it carries copies of one or more numerator elements, which act as part of the primary sex-determination signal, the X:A ratio. The eDp26 duplication is attached to the left tip of the X chromosome in inverted orientation and consequently can be used to generate unstable attached-X chromosomes.     

Localization of Rsf- mutation in Escherichia coli HfrC7]

The contransduction frequency of MAAs, UVs phenotype of Escherichia coli HfrC7 and its 7-51F- derivative with purE markers is found to be 1-2% which indicates that the mutation N 7 is located close to the F integration site in HfrC strain. E. coli strains K-12 7-51F+ and 7-51ColV2+ transfer chromosome markers in the same direction as does HfrC strain. The results suggest the presence of an integrated F fragment (sfa locus) into K-12 7-51F- chromosome.     

The scute (T4) gene acts as a numerator element of the X:a signal that determines the state of activity of sex-lethal in Drosophila.

The ratio of X chromosomes to sets of autosomes (X:A) is the primary genetic signal that determines sex and dosage compensation in Drosophila. The gene Sex-lethal (Sxl) receives this signal and is responsible for the execution of the alternative developmental programmes of males and females. We have found that the scute (T4) gene, which is involved in neurogenesis, also plays a role in the activation of Sxl. The following results suggest that scute (T4) may be a numerator element of the X:A signal: scute (T4) mutations show female-specific lethality. There are female-specific lethal synergistic interactions between sis-a, a previously described numerator element, and mutants for T4. The female lethality is suppressed by SxlM1, a constitutive allele which expresses an active Sxl product independently of the X:A ratio. The Hw685 mutation, which overexpresses T4, is lethal to males with a duplication of sis-a. This lethality is suppressed by either Sxlf1, or the T4 point mutation sc10-1. There are female-specific lethal interactions between sc10-1 and daughter-less (da), a gene needed maternally for Sxl to become active. The sc10-1 mutation masculinizes triploid intersexes.