Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Deficiency of dihydroxy acid dehydratase in the mito-chondria of the iv-1 mutants of Neurospora crassa

The isoleucine-valine requiring mutants of Neurospora crassa which map at the iv-1 locus lack, or have a very low level of activity for, the enzyme dihydroxy acid dehydratase in the mitochondrial fractions derived from them. This enzyme is, however, present in the soluble fractions of the mutant homogenates. The enzyme is present in both mitochondrial and soluble fractions from homogenates of wild-type and from homogenates of iv mutants blocked at other steps in the isoleucine-valine pathway.The work reported here was supported in part by grants GM 12323 and 5TO1-GM-00337-09 from the National Institutes of Health, United States Public Health Service, and by a grant from the Robert A. Welch Foundation.Recipient of Research Career Award 4-K-6-GM-18,383 from the National Institutes of Health, United States Public Health Service.     

Third-Chromosome Mutagen-Sensitive Mutants of DROSOPHILA MELANOGASTER

A total of 34 third chromosomes of Drosophila melanogaster that render homozygous larvae hypersensitive to killing by chemical mutagens have been isolated. Genetic analyses have placed responsible mutations in more than eleven complementation groups. Mutants in three complementation groups are strongly sensitive to methyl methanesulfonate, those in one are sensitive to nitrogen mustard, and mutants in six groups are hypersensitive to both mutagens. Eight of the ten loci mapped fall within 15% of the genetic map that encompasses the centromere of chromosome 3. Mutants from four of the complementation groups are associated with moderate to strong meiotic effects in females. Preliminary biochemical analyses have implicated seven of these loci in DNA metabolism.     

The role of the Golgi complex in the formation of secretion in the test cells of the follicle of the tunicate,Ciona

Summary Electron microscope studies were made on the test cells which comprise a part of the follicular envelope in the ovary of the tunicate Ciona. During development the cells become filled with secretory granules. The Golgi complex is well developed and usually centrally located in the cells. The morphological variations shown and described strongly suggest that the Golgi complex is mainly concerned with the origin of the secretion in these cells.Supported by research grants (GM-9229, 9230) from the National Institute of General Medical Science, United States Public Health Service.This investigation was supported by a Public Health Service Research Career Program Award (1-K3-GM-11, 524) from the National Institute of General Medical Sciences.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

The origin and structure of the secondary coat of the egg of Drosophila melanogaster

Summary The techniques of light and electron microscopy have been employed in a study of the protective coverings of the egg of Drosophila melanogaster. Data obtained during this investigation suggest the involvement of the follicle cells, in the production of one of these coverings and justify its classification as a secondary coat. The secondary coat of D. melanogaster is highly organized and has been divided into three Zones (I, II, IIII). The follicle cells enveloping the oocyte exhibit two phases of secretory activity each involving hypertrophy of the Golgi complex and rough endoplasmic reticulum, and the production of protein and polysaccharide components. The first phase concerns the elaboration of the material which gives rise to the homogeneous lamina referred to as Zone I. The second results in the release of an electron dense component which becomes organized into two laminae separated by struts or pillars; this construction is referred to as Zone II. At the completion of this secretory phase, the follicle cells assume a squamous morphology and a third Zone, composed of a homogeneous substance, appears between the follicle cells and Zone II.This investigation was supported by grant GM-08776 to one of us (EA) from the National Institutes of Health, United States Public Health Service.     

The capacity of the fourth chromosome of Drosophila melanogaster to establish end-to-end contacts with the other chromosomes in salivary-gland cells

When the fourth chromosome of Drosophila melanogaster is attached, as the result of an induced translocation, to 21A in 2L or 60E in 2R, its tip exhibits a marked capacity to pair end-to-end with the tips of the other chromosomes. In each of the translocations, about 59 per cent of the contacts involving the tip of 4 were with the tip of X. If this pairing preference reflects structural similarity, the tip of 4 is much more like the tip of X than that of any other chromosome. The significance of this phenomenon is discussed with respect to the standard pattern of end-to-end association in the Oregon-R wild-type stock that provided the control preparations. In the 4-2L rearrangement, the interaction of chromosome 4 with the tip of 2L (101E with 21A) led to pronounced puffing in subdivisions 21A and B, as was most strikingly manifested when the distal segments of 2L failed to synapse and the homologue of paternal origin showed a large puff whereas that of maternal origin (not carrying the fourth chromosome) remained unpuffed.This study was supported in part by a Research Grant (GM-10499) from the National Institutes of Health, U.S. Public Health Service.     

Genetic variation in mouse salivary amylase rate of synthesis

Heterozygotes from matings of the mouse strains YBR/Cv and C3H/As have about 3 times more YBR-amylase than C3H-amylase in the saliva. The determinant for this quantitative effect is located on linkage group XVI close to or within the structural gene for salivary amylase. The quantitative effect is the result of an increase in the rate of synthesis of YBR-amylase, and the determinant is cis acting. Studies of other mouse strains suggest that regulatory genetic elements may modulate salivary amylase production.This work was supported by the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521).     

Cytological localization of the maternal effect gene, tuh-1, in Drosophila melanogaster

Summary The sex-linked gene, tuh-1, produces a maternal effect that is associated with the tumorous head abnormality in Drosophila melanogaster. With the aid of various known deletions, tuh-1 has been localized to band 20A1-2 on the salivary chromosome map of the X.Work supported by grant GM 18664-01 from the National Institute of Health, U.S. Public Health Service     

Chromosome number in the lizard genus Uta (family Iguanidae)

A chromosome number of 34 (12 macro- and 22 microchromosomes) was found to be characteristic of the bone marrow in 47 animals including males from the species Uta antiquus, and both males and females from the following species and subspecies: Uta stansburiana stansburiana, Uta stansburiana stejnegeri, Uta stansburiana elegans, Uta stansburiana klauberi, Uta stansburiana mannophorus, Uta nolascensis, Uta palmeri, and Uta squamata. — Diploid chromosome numbers of 34 and haploid numbers of 17 were found in the nine testis smears examined. — The presence of a large number of hypodiploid figures in the bone marrow smears is attributed to cell fragmentation and the problem of distinguishing the small microchromosomes. — Series of polyploid figures whose chromosome numbers increased in arithmetic rather than geometric progressions were observed in the testis dry smears. Possible alternatives for the origin of these figures are presented. — Problems encountered in the use of chromosome number as a taxonomic character are discussed.Supported in part by Research Grants GB-366 and GB-5416 from the National Science Foundation, and GM-15361 from the United States Public Health Service.     

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye,ruby-eye-2-J,and maroon

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. Concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney -glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are now known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.This work was supported in part by United States Public Health Service Research Grant GM-19521 and by National Science Foundation Grant PCM77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM07093-03. F. W. was a high school student in the summer program supported by National Science Foundation Grant SP177-26980.     

Theory of hemoglobin facilitated oxygen transport

The transport of oxygen in a hemoglobin-saturated medium is theoretically investigated using classical transport theory. It is found that all the chemical complexes can be expressed as a single function of oxygen pressure. A potential difference together with apH shift is predicted to occur across the medium. This research was supported by the United States Public Health Service Training Grant No. 5-T1-GM-833 from the National Institute of General Medical Sciences. This research was supported by a United States Public Health Service Research Career Program Award 5-K6-GM-18,420 from the National Institute of General Medical Sciences.     

Intragenic mapping of the Neurospora pyrimidine-3 locus by functional deletions

Summary On the basis of complementation between polar and non-polar pyrimidine-3 mutants of Neurospora crassa, a physical linear order of the polar mutants within the locus is derived. The basis of this derivation is that a polar mutant is an intragenic functional deletion extending from the site of the mutation to the translationally distal end of the locus.Supported by Public Health Service Research Grant AI-01462.     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. I. The radioimmunosorbent assay

A radioimmunosorbent technique is described which is capable of independently detecting both isozymes of carbonic anhydrase, CA I and CA II, in concentrations as low as 1 ng/ml. The technique is used to quantitate the different electrophoretic variants of red cell CA I as well as levels of CA II in the pig-tailed macaque, Macaca nemestrina.Supported by U.S. Public Health Service research grant GM-15419.U.S. Public Health Service Predoctoral Trainee (GM-71-14).     

Analysis of chromosome 4 inDrosophila melanogaster. I. Spontaneous and X-ray-induced lethals

An analysis of 17 spontaneous and 37 X-ray-induced lethal mutations on the fourth chromosome ofDrosophila melanogaster has revealed a minimum of 22 loci on this microchromosome capable of mutating to lethality. A few of these loci had been identified earlier by their visible alleles but 16 are new discoveries. Seven of the 22 lethal loci are situated within that proximal section of the right arm of chromosome 4 delimited by theMinute-4 deficiency.Genetic tests indicate that two translocations and five deletions are included among the lethals of X-ray origin. No chromosomal aberrations were found among the spontaneous mutants. Allelism was encountered both within and between lethals from the two groups.Three independent estimates of the total number of lethal loci to be expected on this small autosome are presented. These appraisals are based on (1) the size of theMinute-4 deficiency, (2) the number of bands in salivary chromosome 4, and (3) the frequency of recurrence among the lethals. Considering the uncertainties inherent in each determination, the three estimates (34, 35 and 38) show remarkably good agreement.This investigation was supported in part by U.S. Public Health Service Research Grant GM 11627-01, from the Division of General Medical Sciences.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression

Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFalpha-like molecule Gurken, which controls normal eggshell patterning. We used this easy-to-score eggshell phenotype in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis.     

Analysis of L-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: Complementation for intracellular degradation of the mutant polypeptide

Summary Null and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (-GPDH, NAD⁺ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to providing information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh ⁺. CRM levels appear to be the result of a differential sensitivity to the normal intracellular degradative process and the CRM^- mutants represent hypersensitive alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.This investigation was supported in part by NIH Research Grants No. GM-23617, AG-01739, and by NIH Training Grant No. GM 296. Paper No. 6192 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650     

The origin and fate of secretion in the follicle cells of tunicates

Summary The vacuolization of the outer follicle cells which accompanies maturation of the oocytes of the tunicates Ciona intestinalis and Molgula manhattensis is associated with the dissolution of heterogeneous secretion granules in these cells. The secretion granules, in turn, have a dual origin with one component derived from the endoplasmic reticulum while the other component arises in association with the Golgi complexes. Stages in the morphogenesis of secretion are described.This study was supported by a research grant (GM-09229) and a Career Development Award from the National Institute of General Medical Science, United States Public Health Service.     

Paternal transmission of entire compounds of chromosome two in Drosophila melanogaster

Summary In Drosophila melanogaster, entire compound second chromosomes (2R2L·2L2R) consist of the entire amount of genetic material normally found on separate homologues, as well as significant amounts of heterochromatin derived from the Y chromosome, joined to a single centromere. Genetic analysis demonstrates that information carried upon the Y chromosome influences the rate of transmission of the compound in the male.This work was supported in parts by grants E-501536 and GM-18678 from the National Institutes of Health