Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).     

Deletions of two heat-activated loci in Drosophila melanogaster and their effects on heat-induced protein synthesis.

Deficiencies that delete two heat-induced puffs in Drosophila melanogaster have been isolated. Two deficiencies delete the puff arising from 87C1, and one deletes the two puffs at 87A and 87C1. Homozygotes for these deletions can be recognized by their abnormal, transparent malpighian tubules. The influence on heat-induced protein synthesis of deleting one or both of these puffs has been examined. Unexpectedly, deleting only the 87C1 puff has no apparent effect. Deleting both the 87A and 87C1 puffs eliminates synthesis of a 71,000 dalton protein. We map a coding locus for the 71,000 dalton heat-induced protein to 87A7-B3, a seven band region which also contains a heat-induced puff. The roles of the 87A and 87C1 puffs in coding for heat-induced proteins are discussed in the light of these results.     

Evidence that the enoyl-CoA hydratase bifunctional protein of mouse liver peroxisomes is identical with the 70,000 dalton peroxisomal membrane protein

Peroxisomal enoyl-CoA hydratase was purified from livers of mice treated with di-(2-ethylhexyl)phthalate and its properties compared with those of the 70 kDa protein present in the membranes prepared by carbonate extraction of peroxisomes. The two proteins had identical subunit molecular masses, of about 70,000 daltons. Limited proteolysis of these proteins using the V8 proteinase of S. aureus yielded identical peptide maps, with these peptides crossreacting with antiserum raised against the 70 kDa membrane protein. These data are consistent with the proposal that the peroxisomal 70 kDa membrane protein and the peroxisomal enoyl-CoA hydratase are the same protein.     

Evidence that genes at two loci code for lactate dehydrogenase subunit B in Nycticebus coucang

Lactate dehydrogenase subunits B and A are produced by genes at separate loci. LDH-1, the most anodal of the five isozymes observed after gel electrophoresis, is composed of four B subunits. It has recently been shown that the LDH-1s of most primates are electrophoretically the same. N. coucang (slow loris) is one of the exceptions, possessing an LDH-1 which migrates more slowly than that common to most other primates. We have observed in some members of N. coucang a band at the site of the common primate LDH-1 in addition to the LDH-1 normally present. Since one of the animals in which this observation was made was heterozygous at the LDH B locus, we concluded that in N. coucang two gene loci coding for the B polypeptide are probably present.This investigation was supported in part by contract AF 29 (600)-5587 and NSF grant GB-7426.     

High-resolution mapping of quantitative trait loci for sternopleural bristle number in Drosophila melanogaster.

We have mapped quantitative trait loci (QTL) harboring naturally occurring allelic variation for Drosophila bristle number. Lines with high (H) and low (L) sternopleural bristle number were derived by artificial selection from a large base population. Isogenic H and L sublines were extracted from the selection lines, and populations of X and third chromosome H/L recombinant isogenic lines were constructed in the homozygous low line background. The polymorphic cytological locations of roo transposable elements provided a dense molecular marker map with an average intermarker distance of 4.5 cM. Two X chromosome and six chromosome 3 QTL affecting response to selection for sternopleural bristle number and three X chromosome and three chromosome 3 QTL affecting correlated response in abdominal bristle number were detected using a composite interval mapping method. The average effects of bristle number QTL were moderately large, and some had sex-specific effects. Epistasis between QTL affecting sternopleural bristle number was common, and interaction effects were large. Many of the intervals containing bristle number QTL coincided with those mapped in previous studies. However, resolution of bristle number QTL to the level of genetic loci is not trivial, because the genomic regions containing bristle number QTL often did not contain obvious candidate loci, and results of quantitative complementation tests to mutations at candidate loci affecting adult bristle number were ambiguous.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.     

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.     

Arsenite-induced changes in methylation of the 70,000 dalton heat shock proteins in chicken embryo fibroblasts

Methylated lysyl and arginyl residues are present in the two major heat shock proteins, hsp70A and hsp70B, of chicken embryo fibroblasts. Here, we demonstrate that this methylation can be modulated by sodium arsenite, a chemical that increases the synthesis of hsp70. In particular, in hsp70A the amount of epsilon-N-trimethyl-lysine significantly decreases and the amount of epsilon-N-dimethyl-lysine and epsilon-N-monomethyl-lysine increases, while in hsp70B, the quantity of NG-monomethyl-arginine is reduced fivefold after arsenite treatment. To determine the specificity of these changes in methylation the pool size of S-adenosyl-L-methionine (AdoMet) and the total cellular level of methylated protein was measured. After arsenite treatment, no significant change in AdoMet pool size and the level of protein methylation was observed with the exception of an apparent increase in NG-monomethyl-arginine in total cellular protein. Thus, the arsenite-induced changes in methylation of hsp70 polypeptides are not a generalized phenomenon and may reflect a modulation in the structure or function of these two polypeptides after their induced synthesis by this chemical.     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Deletion mapping of the Aequorea victoria green fluorescent protein

Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2–232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300–500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.     

Robust multipoint simultaneous identical-by-descent mapping for two linked loci

A challenging issue in genetic mapping of complex human diseases is localizing disease susceptibility genes when the genetic effects are small to moderate. There are greater complexities when multiple loci are linked to a chromosomal region. Liang et al. [Hum Hered 2001;51:64-78] proposed a robust multipoint method that can simultaneously estimate both the position of a trait locus and its effect on disease status by using affected sib pairs (ASPs). Based on the framework of generalized estimating equations (GEEs), the estimate and standard error of the position of a trait locus are robust to different genetic models. To utilize other relative pairs collected in pedigree data, Schaid et al. [Am J Hum Genet 2005;76:128-138] extended Liang's method to various types of affected relative pairs (ARPs) by two approaches: unconstrained and constrained methods. However, the above methods are limited to situations in which only one trait locus exists on the chromosome of interest. The mean functions are no longer correctly specified when there are multiple causative loci linked to a chromosomal region. To overcome this, Biernacka et al. [Genet Epidemiol 2005;28:33-47] considered the multipoint methods for ASPs to allow for two linked disease genes. We further generalize the approach to cover other types of ARPs. To reflect realistic situations for complex human diseases, we set modest sizes of genetic effects in our simulation. Our results suggest that several hundred independent pedigrees are needed, and markers with high information, to provide reliable estimates of trait locus positions and their confidence intervals. Bootstrap resampling can correct the downward bias of the robust variance for location estimates. These methods are applied to a prostate cancer linkage study on chromosome 20 and compared with the results for the one-locus model [Am J Hum Genet 2005;76:128-138]. We have implemented the multipoint IBD mapping for one and two linked loci in our software GEEARP, which allows analyses for five general types of ARPs.     

Deficiency mapping of quantitative trait loci affecting longevity in Drosophila melanogaster

In a previous study, sex-specific quantitative trait loci (QTL) affecting adult longevity were mapped by linkage to polymorphic roo transposable element markers, in a population of recombinant inbred lines derived from the Oregon and 2b strains of Drosophila melanogaster. Two life span QTL were each located on chromosomes 2 and 3, within sections 33E-46C and 65D-85F on the cytological map, respectively. We used quantitative deficiency complementation mapping to further resolve the locations of life span QTL within these regions. The Oregon and 2b strains were each crossed to 47 deficiencies spanning cytological regions 32F-44E and 64C-76B, and quantitative failure of the QTL alleles to complement the deficiencies was assessed. We initially detected a minimum of five and four QTL in the chromosome 2 and 3 regions, respectively, illustrating that multiple linked factors contribute to each QTL detected by recombination mapping. The QTL locations inferred from deficiency mapping did not generally correspond to those of candidate genes affecting oxidative and thermal stress or glucose metabolism. The chromosome 2 QTL in the 35B-E region was further resolved to a minimum of three tightly linked QTL, containing six genetically defined loci, 24 genes, and predicted genes that are positional candidates corresponding to life span QTL. This region was also associated with quantitative variation in life span in a sample of 10 genotypes collected from nature. Quantitative deficiency complementation is an efficient method for fine-scale QTL mapping in Drosophila and can be further improved by controlling the background genotype of the strains to be tested.     

Variation in the synthesis of membrane proteins of human T lymphocytes during mitogenic activation: recognition of a 70,000 dalton protein with anti-p23,30 heteroantiserum and a novel 42,000 dalton protein with anti-p44,12 heteroantiserum

Membrane proteins of [35S]methionine-labeled, human T lymphocytes were analyzed by SDS polyacrylamide gradient slab gel electrophoresis and autoradiography each day during a 6-day period of activation with phytohemagglutinin or with concanavalin A. This process was characterized by the novel appearance and limited duration of synthesis of many proteins, in particular of 30, 35, 48, 50, and 55 kilodalton molecules in the early days of blast transformation and subsequently of 120, 125, 135, and 145 kilodalton proteins. The HLAA-A,-B antigens and beta 2-microglobulin, as recognized by anti-p44,12 serum, were synthesized by both resting and mitogen-activated T cells on each day of culture. But, an additional 42-kilodalton protein was recognized with this same antiserum on days 4 and 5 of activation. A 70-kilodalton protein, immunoprecipitated by anti-p23,30 (anti-HLA-protein, immunoprecipitated by anti-p23,30 (anti-HLA-DR) heteroantiserum, was synthesized principally on days 2 and 4 of mitogenic transformation. This molecule was absent from normal, resting T cells, the T cell line, CCRF-CEM, and the B cell line, Raji. In a parallel test, the same anti-p23,30 serum detected the conventional HLA-DR bimolecular glycoprotein complex of 29 and 34 kilodaltons in nonionic detergent solubilized Raji B cell membrane preparations. This study described in detail the molecular changes in the membrane proteins of activated T lymphocytes and included the definition of novel forms of HLA-A,B and HLA-DR associated molecules.     

Crystallographic data for the 9000 dalton wheat non-specific phospholipid transfer protein.

The wheat non-specific phospholipid transfer protein belongs to a family of small proteins sharing a common pattern of four disulphide bridges. Its function in vivo is not known, but it has a high affinity to phospholipids and is involved in phospholipid transfer in vitro. The molecular weight is 9607, and it crystallizes in the space group P2(1) with a = 40.73 A, b = 112.11 A, c = 50.44 A and beta = 106.80 degrees. The crystals diffract to 3 A resolution.     

Patterns of reversion to bobbed condition of magnified bobbed loci in D. melanogaster.

Summary The number of genes coding for the ribosomal RNA (rDNA) can increase in D. melanogaster by means of a process called magnification. In this way, a partial deletion in this locus, termed bobbed, can reach a wild type condition. A newly magnified locus, in turn, reverts to a deficient bobbed condition if it is kept in a phenotypically wild type genotype for several generations. We studied bobbed loci at different magnification steps, analysing their behaviour through the reversion process and the way they carry out a second round of magnification. Results based on the analysis of the reversion process led to the conclusion that magnification consists of a progressive integration into the bobbed locus of free rDNA copies. Moreover, evidence is supplied that the extent of this integration affects the way a reverted locus goes through a second magnification cycle. The extensive characterization of reverted bobbed loci lends substantial support to the extra copies model of rDNA magnification.     

Deficiency mapping of the 93D heat-shock locus in Drosophila melanogaster

The 93D heat shock locus was mapped relative to an overlapping series of deficiencies of the 93D region by three criteria: the ability of the deleted chromosomes to puff at 93D, the ability of the deleted chromosomes to synthesize RNA from the 93D region after a temperature shift and the presence of heat shock RNA sequences at 93D as assayed by in situ hybridization. The results are essentially the same by all three criteria. Chromosomes with deficiencies that did not extend distal to 93D4 puffed and incorporated ³H-uridine after a temperature shift, and were labelled at 93D following in situ hybridization of heat shock RNA from tissue culture cells. All the other deficiency chromosomes tested failed to puff and to incorporate ³H-uridine following a temperature shift and did not show hybridization in this region after in situ hybridization with heat shock RNA. The heat shock locus was mapped to the overlapping region of Df(3R)e ^Gp4and Df(3R)GC14 just outside the inverted region of In(3R)GC23.