Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Suppressible P-element alleles of the vestigial locus in Drosophila melanogaster

Summary A series of P-element insertion mutations at one site in the vestigial (vg) locus was tested for cytotype dependent effects on vg expression. The mutant phenotypes for four P-element vg alleles were suppressed when the alleles were stabilized in the P-cytotype. The suppression was observed whenever repressor-producing P-elements were present in the genome. Genetic and molecular analysis indicated that the suppression is not due to excision or other irreversible alterations of the inserts. The results are consistent with a model in which somatic P-element repressor binding to the ends of P-element inserts can modify the effects of these inserts on target gene expression.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic typical lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Genetic analysis of chromosomal region 97D2-9 of Drosophila melanogaster

The rough homeobox gene of D. melanogaster is required for the correct patterning of the developing eye. The locus maps to cytological location 97D2-5, a region which has not been extensively characterised. As part of our genetic and molecular characterization of rough we carried out an EMS mutagenesis to generate mutants that map to the surrounding region, 97D2-9 which is deleted in Df(3R)ro-XB3. We have generated 1 visible and 13 lethal mutations which, together with the previously described Toll and ms(3)K10 loci, and other unpublished lethals, define nine complementation groups — four lethal, three semi-lethal, one visible and one male-sterile. In addition to rough, one other locus within this region, 1(3)97De, was shown to be required for formation of the normal pattern of photoreceptor cells in the compound eye.The first two authors contributed equally to the work described in this paper     

Molecular organization of master mind, a neurogenic gene of Drosophila melanogaster

Summary The gene master mind (mam) is located in bands 50C23-D1 of the second chromosome of Drosophila melanogaster. mam is one of the neurogenic genes, whose function is necessary for a normal segregation of neural and epidermal lineages during embryonic development. Loss of function of any of the neurogenic genes results in a mis-routeing into neurogenesis of cells that normally would have given rise to epidermis. We describe here the molecular cloning of 198 kb of genomic DNA containing the mam gene. Ten different mam mutations (point mutants and chromosomal aberrations) have been mapped within 45 kb of the genomic walk. One of the mutations, an insertion of a P-element, was originally recovered from a dysgenic cross. Four different wild-type revertants of this mutation were characterized at the molecular level and, although modifications of the insertions were found, in no case was the transposon completely excised. An unusually high number of the repetitive opa sequence, and of an additional previously unknown element, which we have called N repeat, are scattered throughout the 45 kb where the mam mutations map. The functional significance of these repeats is unknown.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Mutant fs(1) 1163 of Drosophila melanogaster alters yolk protein secretion from the fat body

Summary The mutant fs(1) 1163 of Drosophila melanogaster, which was isolated by Gans et al. (1975) is a recessive homozygous female sterile at 18°C and a dominant female — sterile at 29°C. We reported previously that there are reduced quantities of the largest of the three yolk polypeptides in Drosophila melanogaster in the haemolymph and eggs of this mutant at 29°C (Bownes and Hames 1978 a). In this paper we show that the yolk protein defect maps within approximately 2.5 recombination units of the female sterility at 21±2.5 map units on the X-chromosome. The temperature-sensitive period of the yolk protein defect is after emergence. In vitro labelling of fs(1) 1163 ovaries and fat bodies showed that they were able to synthesise yolk polypeptide 1. Interestingly, studies on the proteins present in the various tissues indicate that the fat body tends to accumulate all three yolk polypeptides in the mutant. This phenotype is partially co-dominant in that an effect is seen in heterozygotes as well as homozygotes and is enhanced by increased temperature. This mutant could therefore have a defect (a) in the structural gene for yolk polypeptide 1, (b) in the processing and secretion enzyme systems; (c) in the fat body or all tissues leading to altered secretion properties.Mutants like fs(1) 1163 which alter specific steps in vitellogenesis should be of value for analysing the genetic and biochemical control of the synthesis, transport and sequestering of the yolk polypeptides during oogenesis.     

Isolation of autosomal mutations in Drosophila melanogaster without setting up lines

Summary Mutants of Drosophila melanogaster are being used increasingly for studying different biological mechanisms. However, most attempts to identify new mutations have been restricted to the X-chromosome. It has been very difficult to identify new loci on the autosomes, as recessive mutations have to be made homozygous by setting up independent cultures for each mutagenized chromosome. We introduce a mutagenesis scheme which does not require setting up independent cultures. It uses meiotic recombination in compound autosomes to make recessive mutations homozygous and allows the screening of tens of thousands of mutagenized chromosomes with relatively little effort. In a pilot experiment, we tested about 33,300 chromosomes for temperature-sensitive paralytic mutations. We obtained 62 independent paralytic mutations and a large number of other mutations. Eight out of 25 of the paralytic mutations are on the autosomes. This method makes autosomes, which constitute about 80% of the Drosophila genome, more accessible for mutational analysis of various biological mechanisms.     

Sequence analysis of a yolk protein secretion mutant of Drosophila melanogaster

Summary The female-sterile mutants fs(1) 1163 of Drosophila melanogaster described by Gans et al. (1975) has been characterised as a yolk protein 1 (YP1) secretion mutant (Bownes and Hames 1978b; Bownes and Hodson 1980). We have cloned and sequenced the YP1 gene from this strain, and the strain in which the mutant was induced. One amino acid substitution was found in the predicted polypeptide sequence, an isoleucine to asparagine change at position 92. The sequence of the leader peptide was identical to previously published YP1 sequences. The possible effects of the amino acid change were investigated by computer analysis, which suggests there is no major alteration of secondary structure, but that a hydrophobic region in YP1 is lost in the mutant. This may affect higher order structure.     

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster

A 1194 by open reading frame that codes for a 398 amino acid peptide was cloned from a gt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.     

Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster

There is a rich diversity of paucimannose N-glycans in worms and flies, and these may play a role in the survival of these organisms. Although paucimannose N-glycans are not expressed in vertebrates, complex N-glycans may take over some of the functions of paucimannose N-glycans. Identification of the target proteins of β-1,2-N-acetylglucosaminyltransferase I (GnTI) in worms and flies and elucidation of their functions may thus lead to a better understanding of the role of GnTI-dependent glycoproteins in the survival/longevity of both invertebrates and vertebrates.