Essential Genes in the Hdf6 Region of Chromosome I in Caenorhabditis Elegans

In this paper we describe the analysis of essential genes in the hDf6 region of chromosome I of Caenorhabditis elegans. Nineteen complementation groups have been identified which are required for the growth, survival or fertility of the organism (essential genes). Since ten of these genes were represented by more than one allele, a Poisson calculation predicts a minimum estimate of 25 essential genes in hDf6. The most mutable gene in this region was let-354 with seventeen alleles. An average mutation rate of 5 x 10(-5) mutations/gene/chromosome screened was calculated for an ethyl methanesulfonate dose of 15 mM. Mutations were recovered by screening for lethal mutations using the duplication sDp2 for recovery. Our analysis shows that duplications are very effective for maintenance and mapping of large numbers of lethal mutations. Approximately 600 lethal mutations were mapped in order to identify the 54 that are in the deficiency hDf6. The hDf6 region appears to have a lower proportion of early arresting mutations than other comparably sized regions of the genome.     

Genetic and Phenotypic Analysis of Thirteen Essential Genes in Cytological Interval 22f1-2; 23b1-2 Reveals Novel Genes Required for Neural Development in Drosophila

In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.     

Mutational accessibility of essential genes on chromosome I(left) in Caenorhabditis elegans

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2– a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the occurrence of second-site mutations, thus increasing the probability of recovering single nucleotide substitutions. Treatment of over 32,400 marked chromosomes resulted in 486 analyzed mutations. In this paper, we add 133 previously unidentified let genes, isolated in the EMS screens, and one let gene identified by a γ-ray induced mutation, to our collection of 103 essential genes. We also recovered lethal alleles of genes for which visible mutants already existed. In total, eight deficiencies and alleles of 237 essential genes were identified. Eighty-nine of the previously unidentified let genes are represented by more than one lethal allele. Statistical analysis indicates a minimum estimate of 400 essential genes in the region of chromosome I balanced by sDp2. This region occupies approximately half of chromosome I, and contains over 1135 protein-coding genes predicted from the genomic sequence data. Thus, approximately one-third of the predicted genes are estimated to be essential. Of these approximately 60% are represented by lethal alleles. Less than 2% of the lethal-bearing strains recovered in our analysis, including the eight genetically definable deficiencies, carried more than one lethal mutation. Several screens were used to recover mutations for this analysis. Because all the mutations were isolated using the same balancer, under similar screening conditions, it was possible to compare intervals within the sDp2 region with each other. The fraction of essential genes that present relatively large targets for EMS was highest within the central cluster (dpy-5 to unc-13). Received: 12 July 1999 / Accepted: 6 December 1999     

Genetic analysis of Drosophila melanogaster polytene chromosome region 44D-45F: loci required for viability and fertility

A genetic screen to identify mutations in genes in the 45A region on the right arm of chromosome 2 that are involved in oogenesis in Drosophila was undertaken. Several lethal but no female sterile mutations in the region had previously been identified in screens for P-element insertion or utilizing X rays or EMS as a mutagen. Here we report the identification of EMS-induced mutations in 21 essential loci in the 45D-45F region, including 13 previously unidentified loci. In addition, we isolated three mutant alleles of a newly identified locus required for fertility, sine prole. Mutations in sine prole disrupt spermatogenesis at or before individualization of spermatozoa and cause multiple defects in oogenesis, including inappropriate division of the germline cyst and arrest of oogenesis at stage 4.     

Cytogenetic Analysis of Chromosome Region 73ad of Drosophila Melanogaster

The 73AD salivary chromosome region of Drosophila melanogaster was subjected to mutational analysis in order to (1) generate a collection of chromosome breakpoints that would allow a correlation between the genetic, cytological and molecular maps of the region and (2) define the number and gross organization of complementation groups within this interval. Eighteen complementation groups were defined and mapped to the 73A2-73B7 region, which is comprised of 17 polytene bands. These complementation groups include the previously known scarlet (st), transformer (tra) and Dominant temperature-sensitive lethal-5 (DTS-5) genes, as well as 13 new recessive lethal complementation groups and one male and female sterile locus. One of the newly identified lethal complementation groups corresponds to the molecularly identified abl locus, and another gene is defined by mutant alleles that exhibit an interaction with the abl mutants. We also recovered several mutations in the 73C1-D1.2 interval, representing two lethal complementation groups, one new visible mutant, plucked (plk), and a previously known visible, dark body (db). There is no evidence of a complex of sex determination genes in the region near tra.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations.

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.     

The Doublesex Locus of Drosophila Melanogaster and Its Flanking Regions: A Cytogenetic Analysis

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.     

Genetic analysis of recessive lethal mutations induced by the influenza virus in the X chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M1 + 2. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophila salivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stage of Drosophila. Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10-3C10-11.     

An Extragenic Suppressor of the Mitosis-Defective Bimd6 Mutation of Aspergillus Nidulans Codes for a Chromosome Scaffold Protein

We previously identified a gene, bimD, that functions in chromosome segregation and contains sequences suggesting that it may be a DNA-binding protein. Two conditionally lethal mutations in bimD arrest with aberrant mitotic spindles at restrictive temperature. These spindles have one-third the normal number of microtubules, and the chromosomes never attach to the remaining microtubules. For this reason, we hypothesized that BIMD functioned in chromosome segregation, possibly as a component of the kinetochore. To identify other components that function with bimD, we conducted a screen for extragenic suppressors of the bimD5 and bimD6 mutations. We have isolated seven cold-sensitive extragenic suppressors of bimD6 heat sensitivity that represent three or possibly four separate sud genes. We have cloned one of the suppressor genes by complementation of the cold-sensitive phenotype of the sudA3 mutation. SUDA belongs to the DA-box protein family. DA-box proteins have been shown to function in chromosome structure and segregation. Thus bimD and the sud genes cooperatively function in chromosome segregation in Aspergillus nidulans.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.     

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.     

Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction

Genomic sequences provide powerful new tools in genetic analysis, making it possible to combine classical genetics with genomics to characterize the genes in a particular chromosome region. These approaches have been applied successfully to the euchromatin, but analysis of the heterochromatin has lagged somewhat behind. We describe a combined genetic and bioinformatics approach to the base of the right arm of the Drosophila melanogaster second chromosome, at the boundary between pericentric heterochromatin and euchromatin. We used resources provided by the genome project to derive a physical map of the region, examine gene density, and estimate the number of potential genes. We also carried out a large-scale genetic screen for lethal mutations in the region. We identified new alleles of the known essential genes and also identified mutations in 21 novel loci. Fourteen complementation groups map proximal to the assembled sequence. We used PCR to map the endpoints of several deficiencies and used the same set of deficiencies to order the essential genes, correlating the genetic and physical map. This allowed us to assign two of the complementation groups to particular "computed/curated genes" (CGs), one of which is Nipped-A, which our evidence suggests encodes Drosophila Tra1/TRRAP.     

Characterization of X-Linked Recessive Lethal Mutations Affecting Embryonic Morphogenesis in Drosophila Melanogaster

This study attempted to assay the zygotic contribution of X chromosome genes to the genetic control of embryonic morphogenesis in Drosophila melanogaster. A systematic screen for X-linked genes which affect the morphology of the embryo was undertaken, employing the phenotype of whole mount embryos as the major screening criterion. Of 800 EMS-induced lethal mutations analyzed, only 14% were embryonic lethal, and of these only a minority affected embryonic morphogenesis. By recombination and complementation analyses, the mutations that affected embryonic morphogenesis were sequestered into 26 complementation groups. Fourteen of the loci correspond to genes previously identified in a large-scale screen in which fixed cuticles were examined, and 12 new loci have been identified. Most of the mutations which disrupt embryonic morphology had specific and uniform mutant phenotypes. Mutations were recovered which disrupt major morphogenetic events such as gastrulation, germ band retraction and head involution. No mutations were found which arrest the embryos prior to blastoderm formation. However, a novel class was found, one comprised of mutations which interfere with the development of internal structures but not cuticular structures. Nevertheless, saturation of the X chromosome for genes important for embryonic morphogenesis is probably incomplete.     

Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

A genetic analysis of the cytological region 46C-F containing the Drosophila melanogaster homolog of the jun proto-oncogene

The cytogenetic region 46C-F on the right arm of Drosophila chromosome 2, which contains the homolog of the human jun proto-oncogene, has been genetically mapped and characterized. This project led to the identification and characterization of a Jra (jun-related antigen) mutation, which has been described in detail elsewhere. Three mutagens, EMS, DEB and gamma-rays, were used to isolate 126 lethal lines for this interval. Complementation analysis of the 126 lethal lines identified 29 lethal complementation groups in the region; nine of which have now been correlated with known genes or phenotypes. The region has been subdivided into ten intervals using various small deletions, seven intervals in 46C/D and three intervals in 46E/F. Sixteen P-element lines have been mapped to this interval and are allelic to eight of our complementation groups. The remaining unidentified complementation groups have been analyzed for critical phase, which is when the first observable defect arises and/or when death occurs. There are twelve embryonic lethal groups and seven larval lethal groups. Three lines show visible abnormalities in gut and tracheal development prior to death.     

Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.