Origin and evolution of the transcribed repeated sequences of the Y chromosome lampbrush loops of Drosophila hydei.

The molecular evolution and patterns of conservation of clones from four Y chromosome lampbrush loops of Drosophila hydei were investigated. Each loop contains a discrete family of transcribed repeats that are only slightly conserved even in the hydei subgroup species. Sequencing of clones from the four D. hydei loops indicates that all transcribed repeats evolved from A+T-rich elements of the genome. Evidence is presented that suggests a Y-specific family evolved as a result of the transposition of repeated sequences from an autosomal site to the Y chromosome with the concomitant acquisition of transcriptional activity and loss of non-Y sequences. The results support a structural role for the loops in shaping a spermatocyte-specific nuclear organization. Transcribed heterochomatic sequences could play a similar role in nuclear organization in many cell types.

The zeste-white interaction: induction and genetic analysis of a novel class of zeste alleles.

The recovery and analysis of a novel class of zeste mutations is described. z and z do not require two w genes for their expression unless the z gene is present. Analysis of genetic interactions among zeste alleles proved that z mutations are strong antagonists of the z gene product. z is readily reverted by X-rays or by ethylmethane sulphonate to a range of intermediate zeste alleles and thus it is considered to be an insertional mutation. We suggest that z and op mutations arose as a result of insertions in a presumptive control region, and two alternative models for the structure of the zeste locus are evaluated.

Sequence replication and banding organization in the polytene chromosomes of Drosophila melanogaster.

The relative proportions of cloned DNA fragments from all known hierarchies of sequence organization in polytene and diploid chromosomes were compared. It was found that unique sequences of varying sizes and chromosomal locations are equally replicated in salivary gland chromosomes. Sequences of euchromatic polydisperse gene families are also replicated proportionately in polytene and diploid tissues. Perhaps the most significant finding is that the histone gene repeats, despite their normal banding organization, are under-replicated in the polytene chromosome of Drosophila melanogaster. However, the clustered and well-banded 5S genes are most likely equally replicated. It is therefore concluded that differential sequence replication plays no apparent role in either the assembly or morphology of a band; and likewise, the assembly of polytenic DNA into band units is not affected by either the local abundancy or arrangement of middle repetitive sequences. The likelihood that the clustered arrangement is an important factor in the selection of sequences for under-replication is discussed.

DNA clones and RNA transcripts of four lampbrush loops from the Y chromosome of Drosophila hydei.

Drosophila hydei clones representing transcribed middle-repetitive sequences from four of six major lampbrush loops of the Y chromosome were isolated. Sequences homologous to each clone are clustered in a particular locus on the Y chromosome, but additional euchromatic sites were found for one of the transcribed clones. In situ hybridization to lampbrush-loops RNA permitted the identification of clones homologous with the two "nooses" loops on YS and with the "clubs" and "tubular ribbons" on the YL arm. Loop-specific nuclear RNA molecules range in size from 10S to 60S. Loop RNA is accumulated in the nucleus and remains attached to the loops during the course of primary spermatocyte growth. It disappears, however, along with the loop structures, during the first meiotic prophase. The structure and function of the Y chromosome and its lampbrush loops are briefly considered in the light of these findings.

Heterochromatin markers: a search for heterochromatin specific middle repetitive sequences in Drosophila.

We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X-and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.

Heterochromatin markers: arrangement of obligatory heterochromatin, histone genes and multisite gene families in the interphase nucleus of D. melanogaster.

Localization, as detected by in situ hybridization, of major heterochromatic blocks in interphase nuclei of larval brain and imaginal discs is reported. We conclude that the position of heterochromatic regions in interphase nuclei is correlated with their respective position in metaphase chromosomes and hence, independent of sequence recognition. Furthermore, chromocentral associations of X-, Y- or autosomal-based heterochromatin are not formed in these cells. Homologues do align in close proximity, but heterochromatin plays no role in this arrangement. Heterochromatin, and probably nucleoli, establish their membrane links in situ, and have no prefixed recognition sites. The most intimate association between homologous repetitive sequences was found in the histone locus, but no tendency for clustering was found among loci of multisite euchromatic gene families.

Genetic identification of dominant overproducing mutations: the Beadex gene.

A model system for the identification of presumptive overproducing mutations from among visible dominant mutations in D. melanogaster is described. An overproducing mutation is expected if a dominant mutation is readily reverted by gene deletion and if gene deletions suppress the expression of the original dominant mutation in flies heterozygous for the deletion. The Beadex (1:59.4) mutations are shown to satisfy these requirements, since a Bx dominant mutations is reverted by induced deletion [Df(Bx)/+) is wild type], and is also suppressed in trans by such a deletion [(Bx/Df)Bx) is wild type]. In addition, all 13 mutations recovered as Bx reversions or suppressors were associated with recessive held up (hdp) mutations allelic inter se, but not allelic to any known hdp gene. One such hdp mutations does not function as an independent dominant suppressor of Bx, is not always associated with Bx deletion, and in the latter situation is readily separable from Bx. We suggest that it functions as a Bx deletion, and may therefore represent the structural gene which is cis-regulated by the overproducing Bx mutations.

The role of X-linked lethal and viable male-sterile mutations in male gametogenesis of Drosophila melanogaster: genetic analysis.

The possibility that viable male-sterile mutations occur in vital genes and the role played by lethal mutations and viable male-steriles in male gametogenesis were studied. Five sterile loci were identified among the 30 most proximal vital loci of the X-chromosome and two of them were shown to be allelic with lethal mutations. Fertility test on gynanders for nonautonomous lethal mutations proved that vital genes operate autonomously in male gonads, independently of their effect on somatic tissues. Fertility tests of ts lethals, shifted to the nonpermissive temperature after the TSP, showed that 40% of vital genes function in male gonads. It is further shown that about the same proportion of vital genes is operating in female gonads and that the two groups overlap by about 70%. The role of viable and lethal male gametogenesis is discussed in detail.

Fine-Structure Analysis and Genetic Organization at the Base of the X Chromosome in DROSOPHILA MELANOGASTER.

Genetic organization at the base of the X chromosome was studied through the analysis of X-ray-induced deficiencies. Deficiencies were recovered so as to have a preselected right end "anchored" in the centric heterochromatin to the right of the su(f) locus. "Free" ends of deficiencies occurred at any of 22 intervals in Section 20 and in the proximal portion of Section 19 of Bridges' (1938) polytene chromosome map. The distribution of 130 such free ends of deficiencies induced in normal, In(1)sc( 8), and In(1)w(m4) chromosomes suggests that on the single section level, genes are flanked by "hot" or "cold" sites for X-ray-induced breaks, and that occurrence of the hot spots is dependent on their interaction with the fixed-end sites in the centric heterochromatin. In the light of these results, it is argued that long heterochromatic sequences separate the relatively few genes in Section 20, and thus endow it with several characteristics typical of heterochromatic regions. Section 20 is considered to be a transition region between the mostly heterochromatic and mostly euchromatic regions of the X chromosome; the differences between them are suggested as being merely quantitative.

Characterisation of male meiotic-sterile mutations in drosophila melanogaster. The genetic control of meiotic divisions and gametogenesis.

Male meiotic sterile mutations were selected among X-linked male-steriles by detection of micronuclei in early spermatids. Despite severe defects in the 1st or 2nd meiotic spindles in all mutants, no effect on mitosis was observed. Various features of spindle structure, chromosome segregation, and centriole movements were compared in seven meitoic steriles and in XO males. Chromosome behaviour and centriole movement were always affected concomitantly, and were both shown to be genetically independent of "centre" formation in the meiotic spindles. Precocious and delayed centromere separation was observed in the various mutants in both divisions, and similarly attributed to basic spindle lesions rather than chromosome defects. Attachment of the centriole body to the membrane of the spermatid nucleus was normal only in mutants where second division nuclei were formed. The role of the centriole body was shown to be independent of membrane attachment.--The phenomena observed in this study were discussed mainly with regard to genetic interdependence of morphogenetic processes during male meiosis. A common base for the pleiotropic defects of meiotic steriles and XO males is suggested, and the genetic control of meiosis is re-evaluated in the light of comparison with fertile meiotic mutants.

Genes controlling chromosome activity; the role of genes blocking Y-lampbrush loop propagation.

Characterization of two more X-linked Y-affecting mutations in D. hydei is presented; ms(1) XL24 is less extreme than the previously described mutation, ms(1)XL2, but its general effect on the propagation of Y-lampbrush loops is similar qualitatively; ms(1)XL24 is located at the distal end of the X-chromosome and associated with a suppressor-of-white phenotype. The other X-linked sterile, ms(1)XL4, is the least extreme of the three mutations examined to date. It is recessive, located ca 2 c.o. units to the right of white, and causes different patterns of Y loop response. -- From a comparison of the effects of the three single mutations, as well as from the analysis of the various double-sterile combinations, we surmise that: 1) if specific activator genes for the Y-lampbrush loops do exist, they are not likely to be located on the X-chromosome of Drosophila; 2) every lampbrush loop is composed of several functional repeats, each of which operates when unfolded; 3) the Y-affecting mutations arrest preferentially, but not exclusively or specifically Y-lampbrush loop propagation probably indirectly through their independent metabolic effects. Since the mutations are not specific for germ line cells, and exhibit various pleiotropic effects, especially with regard to viability, they should be considered tissue conditional lethals.

Differential sensitivities and the target of heat-induced recombination at the base of the X chromosome of Drosophila melanogaster.

The effect of heat on two small adjacent segments at the base of the X chromosome was examined. Recombination in the two segments delimited by recessive lethals was measured after treatment with 30 degree and 34 degree. Both segments were sensitive at 30 degree, while only the proximal one responded to 34 degree treatment. When the same segments were studied in structural heterozygotes (for deletions) the relative increase in recombination was greater, suggesting that heat exerts its effect on the "pairing" rather than the "exchange" components of crossing over. The effect of c (3) G/+ on the same segments in both homozygous and heterozygous structurals was studied. The results indicate that this meiotic mutant mediates its effect on a step different than that affected by heat.

The role of X-chromosome inactivation during spermatogenesis (Drosophila-allocycly-chromosome evolution-male sterility-dosage compensation).

Inactivation of the single X chromosome in the primary spermatocytes of species with heterogametic males is postulated as a basic control mechanism on the chromosomal level that is required for normal spermatogenesis. This view is supported by (a) cytological observations of X-chromosome allocycly in the primary spermatocytes of all male-heterogametic organisms that were adequately examined, (b) autoradiographic evidence of early cessation of transcription by the X chromosome in the mouse and three species of grasshopper, and (c) the male sterility of animals with certain X-chromosome rearrangements that cannot be attributed to misfunction of specific genes. X-chromosome inactivation during spermatogenesis is proposed as the ideal system for studies of genetic control at the chromosomal level.