Hypersensitivity of Drosophila mei-41 mutants to hydroxyurea is associated with reduced mitotic chromosome stability

6 mutant alleles of the mei-41 locus in Drosophila melanogaster are shown to cause hypersensitivity to hydroxyurea in larvae. The strength of that sensitivity is directly correlated with the influence of the mutant alleles on meiosis in that: alleles exhibiting a strong meiotic effect (mei-41D2, mei-41D5, mei-41D7) are highly sensitive; alleles with negligible meiotic effects (mei-41(104)D1, mei-41(104)D2) are moderately sensitive and an allele which expresses meiotic effects only under restricted conditions (mei-41D9) has an intermediate sensitivity. This sensitivity is not a general feature of strong postreplication repair-deficient mutants, because mutants with that phenotype from other loci do not exhibit sensitivity (mus(2)205A1, mus(3)302D1, mus(3)310D1). The observed lethality is not due to hypersensitivity of DNA synthesis in mei-41 larvae to hydroxyurea as assayed by tritiated thymidine incorporation. Lethality is, however, potentially attributable to an abnormal enhancement of chromosomal aberrations by hydroxyurea in mutant mei-41 larvae. Both in vivo and in vitro exposure of neuroblast cells to hydroxyurea results in an increase in 3 types of aberrations which is several fold higher in mei-41 tissue. Since hydroxyurea disrupts DNA synthesis, these results further implicate the mei-41 locus in DNA metabolism and provide an additional tool for an elucidation of its function. The possible existence of additional genes of this nature is suggested by a more modest sensitivity to hydroxyurea which has been detected in two stocks carrying mutagen-sensitive alleles of alternate genes.     

The single copy gene coding for human α1 (IV) procollagen is located at the terminal end of the long arm of chromosome 13

Summary Using dual-laser sorted chromosomes and spot-blot analysis, we have previously assigned genomic DNA sequences coding for human 1(IV) procollagen to chromosome 13 (Pihlajaniemi et al. 1985). By in situ hybridization to normal chromosomes and chromosomes with 13q deletions, we now report the localization of this gene to the terminal end of the long arm of chromosome 13. In addition, Southern and slot blot hybridization analysis clearly show that these genomic sequences are present only once per haploid genome.     

Mannoproteins from the cell wall of Kluyveromyces lactis

Abstract Wall mannoproteins from Kluyveromyces lactis have been solubilised by treatment of cell walls with sodium dodecyl sulphate (SDS) or zymolyase. While the former reagent liberates a large number of molecular species, zymolyase preferentially releases a high-molecular-weight material that is sensitive to endo- β - N -acetylglucosaminidase H, and a 29-kDa molecule that reacts with the antiserum raised against a similar species from walls of Saccharomyces cerevisiae . In contrast with observations on isolated walls of S. cerevisiae , dithiothreitol pretreatment of K. lactis walls does not enhance the effect of zymolyase upon mannoprotein release. However, the action of thiol agents is still necessary to obtain protoplasts by zymolyase digestion from K. lactis whole cells.     

Reproductive performance of postpartum beef cows after short-term calf separation and dietary energy and protein supplementation

Beef cows (n = 294) calving between November and April in six states were used to evaluate the effects of postpartum diet and calf separation on body weight, body condition score (BCS), reproductive performance, and weaning weight of calves. In each state, half of the 48 cows that calved during 60 d were group fed an additional 4.5 kg of a 20% crude protein supplement daily for 28 d starting an average of 30 d post partum (flush). Calves were separated from half of the flush and half of the nonflush cows for 48 h at 14 and 28 d after the beginning of flush. Progesterone was quantified in plasma samples obtained weekly during a 56-d breeding period to assess ovarian luteal activity. The breeding period started at the first calf separation. BCS ranged from 3.3 to 5.6 among states (on a scale of 1 to 9) at the start of the flush but was similar for treatments within a state. There was a state x flush (P < 0.008) effect on body weight at the end of the flush period. Weaning weights were influenced by state x separation x flush (P < 0.06) and were greatest for flush nonseparated calves in five of six states. There were state x flush (P < 0.08) and separation (P < 0.04) effects on ovarian luteal activity at the start of the breeding period. Flush and separation tended to increase ovarian luteal activity. During the breeding period, ovarian luteal activity was influenced only by state but there was a state x separation x flush effect (P < 0.001) on the number of weeks post partum to onset of ovarian luteal activity. Conception rate and days postpartum to conception were not influenced by either separation or flush but were affected by state (P < 0.001). These data indicate that flushing may increase weaning weights of calves and calf separation may hasten the onset of postpartum ovarian luteal activity, but conception rate and days postpartum to conception for cows in thin to moderate body condition were not influenced by the calf separation or flushing treatments.     

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).     

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.     

Regional chromosomal assignment of the human glucocorticoid receptor gene to 5q31

Summary The gene for the human glucocorticoid receptor, previously mapped to chromosome 5, has been further localised to 5q31 by in situ hybridisation using a biotinylated 4.3-kb cDNA probe.     

Recurrences of trisomy 18 and trisomy 13 after trisomy 21

Summary Between 40 years and 43 years of age, a woman had three consecutive pregnancies with different prenatally diagnosed autosomal trisomies. This is compatible with the view that the predisposition to non-disjunction is not chromosome-specific.     

Isolation and characterization of a human variable copy number tandem repeat at Xcen-p11.22

A subclone (M27B) has been isolated from a cosmid randomly selected from a library enriched for human X-chromosomal material. The subclone is extensively single-copy sequence, but also contains three complete copies and one partial copy of a 26-bp repeat, within which exists an inverted repeat having the potential to form a cruciform loop structure. Genomic sequences in this repeat region are apparently refractory to cloning, and rearrangements occurring during this process result in deletions. M27B detects multiple X-linked restriction fragments for a wide range of enzymes including MspI and HpaII. We have assigned the locus recognized by the probe (DXS255) to Xcen-Xp11.4 by mapping with a somatic cell hybrid panel and further refined its localization to Xp11.22 by in situ hybridization. The repeat sequence that is presumed to be responsible for the hypervariability observed does not show close similarity to other variable copy number tandem repeats described.     

Cloning and characterization of a phospholipase gene from Erwinia chrysanthemi EC16.

A single gene (plcA) was cloned from a cosmid library of Erwinia chrysanthemi EC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39kDa. The coding region was G+C-rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed in Escherichia coli cells, the plcA gene directed production of a c. 39kDa protein that was largely localized in the periplasm, but its N-terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild-type bacterium, an E. chrysanthemi EC16 marker exchange mutant of the plcA gene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum stems.