Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.     

Different genetic components in the Ethiopian population, identified by mtDNA and Y-chromosome polymorphisms

Structural equation modeling procedures were used to examine relationships among several war zone stressor dimensions, resilience-recovery factors, and post-traumatic stress disorder symptoms in a national sample of 1,632 Vietnam veterans (26% women and 74% men). A 9-factor measurement model was specified on a mixed-gender subsample of the data and then replicated on separate subsamples of female and male veterans. For both genders, the structural models supported strong mediation effects for the intrapersonal resource characteristic of hardiness, postwar structural and functional social support, and additional negative life events in the postwar period. Support for moderator effects or buffering in terms of interactions between war zone stressor level and resilience-recovery factors was minimal.     

HLA, Gm, and Km polymorphisms and immunity to infectious diseases in South Amerinds

Isolated South Amerinds, a population at very high risk from infectious disease, mount good immune responses to pneumococcal polysaccharides, viral antigens and other immunogens. No unusual immunoglobulin allotype or HLA antigen, which might explain the high risk from infectious disease, was found among them. Responses are examined in relation to the immunogenetic markers that are most prevalent. Amerinds with Gm 1,2,17,21 responded less well than persons without this haplotype to 10 of 12 pneumococcal polysaccharides, and those who were homozygous at the HLA class I loci responded less well to viral antigens. However, these differences are not strong and there are few such findings relative to the number of possibilities examined. The most distinctive immunogenetic characteristic of these populations is their low level of polymorphism at all tested loci. Their susceptibility to infectious agents can be attributed to this genetic uniformity and a consequent ability of pathogens to adapt to the population.     

Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: An objective of therapeutic intervention after cerebral ischemia is to promote improved functional outcome. Improved outcome may be associated with a reduction of the volume of cerebral infarction and the promotion of cerebral plasticity. In the developing brain, neuronal growth is concomitant with expression of particular proteins, including microtubule-associated protein 2 (MAP-2), growth-associated protein 43 (GAP-43), and cyclin D1. In the present study we measured the expression of select proteins associated with neurite damage and plasticity (MAP-2 and GAP-43) as well as cell cycle (cyclin D1) after induction of focal cerebral ischemia in the rat. METHODS: Brains from rats (n=28) subjected to 2 hours of middle cerebral artery occlusion and 6 hours, 12 hours, and 2, 7, 14, 21, and 28 days (n=4 per time point) of reperfusion and control sham-operated (n=3) and normal (n=2) rats were processed by immunohistochemistry with antibodies raised against MAP-2, GAP-43, and cyclin D1. Double staining of these proteins for cellular colocalization was also performed. RESULTS: Loss of immunoreactivity of both MAP-2 and GAP-43 was observed in most damaged neurons in the ischemic core. In contrast, MAP-2, GAP-43, and cyclin D1 were selectively increased in morphologically intact or altered neurons localized to the ischemic core at an early stage (eg, 6 hours) of reperfusion and in the boundary zone to the ischemic core (penumbra) during longer reperfusion times. CONCLUSIONS: The selective expressions of the neuronal structural proteins (MAP-2 in dendrites and GAP-43 in axons) and the cyclin D1 cell cycle protein in neurons observed in the boundary zone to the ischemic core are suggestive of compensatory and repair mechanisms in ischemia-damaged neurons after transient focal cerebral ischemia.     

Alteration of protein metabolism in individual, identified neurons from Aplysia

A search for control mechanisms governing protein metabolism in neurons from Aplysia californica has uncovered two examples of altered patterns of newly synthesized proteins: (1) The pattern of newly synthesized proteins in the R2 neuron is altered when protein synthesis occurs at elevated temperatures (22-30 degrees C as compared with 13-15 degrees C). (2) The processing of newly synthesized 12,000 dalton (12k) material to 6-9,000 dalton (6-9k) size in the R15 neuron (Strumwasser, F. and Wilson, D.F. [1976], J. Gen. Physiol., in press) can be blocked by certain ion replacements. If acetate replaces chloride in the incubation medium during the synthesis of 12k material, an early step in the processing, prior to the actual breakdown of 12k material, is blocked. Experiments with RNA-synthesis inhibitors indicate that none of the mRNAs which code for abundantly synthesized protein species in the R2 or R15 neurons have short (less than 4 hr) half-lives. This result has implications for an earlier report of regulation of protein synthesis in the R15 neuron.     

DNA bending by a phantom protein

BACKGROUND: Despite its stiffness, duplex DNA is extensively bent and folded during packaging and gene expression in biological systems. Modulation of the electrostatic repulsion between phosphates in the DNA backbone may be important in the bending of DNA by proteins. Here, we analyze the shape of DNA molecules that have been modified chemically to mimic the electrostatic consequences of a bound protein. RESULTS: We have simulated salt bridges between DNA phosphates and cationic amino acid sidechains of a phantom protein by tethering ammonium cations to one face of the DNA helix. Tethered ammonium cations, but not neutral acetylated controls, induce DNA to bend toward its neutralized surface. CONCLUSIONS: The shape of DNA molecules bearing a laterally-asymmetric distribution of tethered cations agrees qualitatively with theoretical predictions and with results previously obtained using neutral phosphate analogs. These data suggest principles that might be applied to the design of artificial DNA-bending proteins.     

HLA class II DRB1, DQA1 and DQB1 polymorphisms in the Polish population from Wielkopolska

HLA DRB1, DQA1 and DQB1 alleles were determined by DNA PCR-SSO typing in a sample of 99 individuals originating from Wielkopolska (midwestern Poland). A high number of alleles (38 DRB1, 8 DQA1 and 14 DQB1) was detected at each locus, many of them presenting notable frequencies in this population. The three HLA loci are thus characterized by very high heterozygosity levels (93% for DRB1, 85% for DQA1, and 88% for DQB1), which confirms the results found for other European populations. A total of 6 DRB1-DQA1-DQB1 haplotypes are detected with an estimated frequency higher than 5%, namely, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*0701-DQA1*0201-DQB1*0201, DRB1*0101-DQA1*0101-DQB1*0501, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*03011-DQA1*0501-DQB1*0201, and DRB1*1301-DQA1*0103-DQB1*0603. A genetic distance analysis between the Polish and other world populations tested for HLA class II indicates that the Wielkopolska community is close to geographically close, rather than linguistically related populations from Europe. More generally, a good agreement between genetics and geography is found for DRB1 and DQB1 polymorphisms in Europe, suggesting that these two loci are highly informative for assessing historical relationships among humans.     

HLA class I and II polymorphisms and trachomatous scarring in a Chlamydia trachomatis-endemic population

Immune responses to Chlamydia trachomatis contribute to protection from infection and to immunopathologic disease. To test whether subjects' HLA class I (A, B, and Cw) or class II (DRbeta1 and DQbeta1) types influence risk of trachomatous scarring from chronic infection with C trachomatis, 153 cases and pair-matched controls in Gambia were studied. No HLA type was associated with protection from scarring, indicating that protective immune responses are not limited to only one or a few HLA-restricted epitopes in C. trachomatis antigens. One class I antigen, HLA-A28, was significantly more common among cases than controls (25.8% vs. 15.9%, respectively; McNemar's odds ratio [OR], 1.88; 95% confidence interval [CI] = 1.01-3.49; P = .046). In DNA subtyping of the A28 specificity, the A*6801 allele was equally common among cases and controls, but the A*6802 allele was significantly overrepresented among cases (McNemar's OR, 3.14; 95% CI = 1.32-7.44; P = .009). This association may be due to an immunopathologic HLA-A*6802-restricted cytotoxic T lymphocyte response.     

DNA amplification polymorphisms of Mucor piriformis

OBJECTIVE: The aim was to determine the chorionic and amniotic types in multifetal pregnancies with transvaginal ultrasonography at very early stage of gestation. STUDY DESIGN: Twenty-one spontaneous multifetal pregnancies were scanned transvaginally before 8 weeks' gestation (four of them from 4th week). The chorionic and amniotic type was determined ultrasonographically. All twin gestations had postpartum pathologic evaluation of the placenta and histologic determination of the chorionic and amniotic type. RESULTS: Ultrasonographic evaluation of the 21 pregnancies demonstrated 20 twin and 1 triplet gestation. Four of the twin pregnancies were monochorionic-diamniotic. Triplet was monochorionic-triamniotic (spontaneously aborted in 8th week of gestation). In all 20 twin pregnancies, transvaginal ultrasonography correctly predicted the chorionic and amniotic type before 8 weeks of gestation. CONCLUSION: Transvaginal ultrasonography allows a reliable, simple and rapid determination; the dichorionic twin pregnancy in 4 weeks, monochorionic in 5 weeks, and differentiation of mono- or diamniotic in 7 weeks of gestation.     

Genetics of salivary protein polymorphisms

Human salivary PRPs are determined by six closely linked genes on chromosome 12p13.2. The many PRPs show complex electrophoretic patterns that differ between individuals and reflect numerous genetic polymorphisms. Frequent length and null polymorphisms are common among PRPs. Common themes emerge as a background for these PRP polymorphisms. First, posttranslational proteolysis occurs with double-banded patterns among acidic PRPs and the generation of numerous basic PRPs derived from precursor proteins. Specific mutations may interfere with proteolysis, preventing generation of double-banded acidic PRPs (as with the Pa protein) or of small basic PRPs from precursor proteins (as with Pm proteins). Second, single cysteine substitutions in PRPs (Pa from PRH1 and G1 8 from PRB3) may lead to disulfide bonded homodimers as well as heterodimers with salivary peroxidase. Third, frequent homologous and unequal crossing-over within the PRP gene cluster leads to frequent protein size-variants (intragenic events as with the G1 protein variants) and the generation of the PRB2/1 fusion gene (intergenic event) with deletion of the PRB1 coding region and absence of multiple PRB1 coded proteins (Ps, Pm, Pe) in PRB2/1 homozygotes. Fourth, null mutations may also be produced (as with PsO and G1 0) by single nucleotide changes.     

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.     

Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein

In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA. We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay. RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact. Normally, neither UmuD nor UmuD' shows any affinity for DNA. In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA. RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA. These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA.     

Spinocerebellar ataxia and HLA linkage: risk prediction by HLA typing

To determine the possibility of genetic linkage of spinocerebellar ataxia with the histocompatibility loci, we performed HLA typing and linkage analysis on 19 members of a kindred in which spinocerebellar ataxia was segregating in an autosomal dominant inheritance pattern. The ataxia locus was located on chromosome 6 at 12-cM distance from the HLA complex with lod score of 3.15 (odds is greater than 1400:1 favoring linkage over chance findings). Thus, the presence of the ataxia gene in members of this kindred at risk can be predicted with about 90 per cent accuracy by means of HLA typing in informative matings.     

Cholangiocyte-specific rat liver proteins identified by establishment of a two-dimensional gel protein database

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.     

Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila

Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan. These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain). Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3. They map to all five chromosomes and appear to be evenly distributed throughout the genome. Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5. The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.     

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.