Molecular characterization of de novo secondary trisomy 13.

Unbalanced Robertsonian translocations are a significant cause of mental retardation and fetal wastage. The majority of homologous rearrangements of chromosome 21 in Down syndrome have been shown to be isochromosomes. Aside from chromosome 21, very little is known about other acrocentric homologous rearrangements. In this study, four cases of de novo secondary trisomy 13 are presented. FISH using alpha-satellite sequences, rDNA, and a pTRI-6 satellite I sequence specific to the short arm of chromosome 13 showed all four rearrangements to be dicentric and apparently devoid of ribosomal genes. Three of four rearrangements retained the pTRI-6 satellite I sequence. Case 1 was the exception, showing a deletion of this sequence in the rearrangement, although both parental chromosomes 13 had strong positive hybridization signals. Eleven microsatellite markers from chromosome 13 were also used to characterize the rearrangements. Of the four possible outcomes, one maternal Robertsonian translocation, two paternal isochromosomes, and one maternal isochromosome were observed. A double recombination was observed in the maternally derived rob(13q13q). No recombination events were detected in any isochromosome. The parental origins and molecular chromosomal structure of these cases are compared with previous studies of de novo acrocentric rearrangements.     

De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli.

dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP.     

Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.

Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.     

Risk of breast cancer in relation to the interval since last full term pregnancy.

OBJECTIVE--To examine whether the risk of breast cancer is increased by a recent term pregnancy. DESIGN--Population based case-control study. SETTING--Eight areas in the United States. SUBJECTS--Cases were 2279 multiparous women residents of the eight areas aged 25-49 who were diagnosed as having breast cancer during 1980-2. Controls were 2357 multiparous women selected from the same areas by random digit dialing. MAIN OUTCOME MEASURE--Relative risk of developing breast cancer according to the time interval since last full term pregnancy. RESULTS--The distribution of intervals since the last term pregnancy was similar in cases and controls. Adjusted for age, parity, and age at first term pregnancy, the odds ratios observed for categories of years since the last full term pregnancy were: 0-2 years, odds ratio 1.16 (95% confidence interval 0.84 to 1.59); 3-6 years, odds ratio 1.21 (0.95 to 1.54); 7-9 years, odds ratio 1.04 (0.84 to 1.38); > or = 10 years, odds ratio 1.00 (reference). CONCLUSIONS--Among multiparous women aged 25-49 years there was no association between the risk of breast cancer and the time interval since the last full term pregnancy.     

Adult emergence phenology in checkerspot butterflies: the effects of macroclimate,topoclimate, and population history

The prediction of adult emergence times in insect populations can be greatly complicated by microclimatic gradients, especially in circumstances where distributions of juveniles along those gradients vary from year to year. To investigate adult emergence patterns in topographically heterogeneous habitats, we built a model of postdiapause development of the Bay checkerspot butterfly, Euphydryas editha bayensis. The model uses slope-specific insolation as the rate-controlling variable, and accounts for both solar exposure of the habitat and cloud cover. Instar-specific larval mass gains per unit of insolation were determined from mark-recapture experiments. A small correction for daily low temperatures was used to calibrate the model to five years of field data on larval mass. The model predicted mean mass of 90% of larval samples within 4 clear days over a 70–120 day growing season. The magnitude of spatial variation in emergence times across habitat slopes is greater than annual variation in emergence times due to yearly weather conditions. Historical variation (yearly shifts in larval distributions across slopes) is an important determinant of mean population emergence dates. All of these factors need to be considered in understanding adult emergence phenology in this butterfly and in other insects inhabiting heterogeneous thermal environments. Such an understanding can be useful in managing insect populations for both pest control and conservation.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.     

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.