Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5 truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Unigenic and multigenicI region control of the immune responses of mice to the GAT10 and GLΦ-GLT terpolymers

Recombinant strains of mice with known alleles in theI region of theH-2 complex were used to map theH-2 linked immune response genes controlling responsiveness to random terpolymers GAT¹⁰ and GL. TheIr-GAT gene was mapped to either theIA orIB subregions. In contrast, data obtained in the GL-GLT system indicated multigenic control. The responsiveness of the B10.A(3R), B10.A(5R), and B10.S(9R) recombinants indicated that one immune response gene,IrGL-GLT _A, mapped to the right ofIB, i.e., in theIC subregion. The nonresponsiveness of the B10.A(1R), B10.A(2R), B10.M(17R), and AQR mice having responderIC ^d alleles butIA ^k-IB ^k nonresponder alleles and the positive response of a (C57BL/6 × A/J)F₁ hybrid derived from two nonresponder parental strains indicated the presence of a second gene inIA-IB subregions,Ir-GL-GLT _B. The interaction between these two genes, each present in a differentI subregion, controls the immune response.     

Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9. Address correspondence and offprint requests to: R. D. Campbell.     

Microsatellite genotyping of Chromosome 14q13.2-14q13 in the vicinity of proteasomal gene<Emphasis Type="Italic"> PSMA6</Emphasis> and association with Graves’ disease in the Latvian population

The 270-kb Chromosome 14q13.2-14q13 region harboring the proteasomal alpha subunit 6 gene PSMA6 was analyzed for polymorphism of five microsatellite repeats in cases/controls and association with Graves disease. Four novel microsatellite markers were localized to the 14q13.2 region upstream of PSMA6. Dinucleotide repeats HSMS801, HSMS702, HSMS701 were identified in two introns of the gene KIAA0391; the most upstream trinucleotide HSMS602 marker was found in an intron of the C14orf24 gene. A polymorphism study performed on the Latvian population revealed 13 and 14 alleles for HSMS801 and HSMS702, respectively, seven alleles for HSMS701, and four alleles for HSMS602. Heterozygosity analysis revealed that all the four markers obey Hardy-Weinberg distribution. The previously described HSMS006 marker, represented by 12 alleles, is localized in intron 6 of the PSMA6 gene. No significant differences were observed between patients and controls in allele distribution of the HSMS702 and HSMS701 microsatellite repeats. However, the allele frequencies of HSMS006 and HSMS801 were significantly different between Graves disease and control subjects. The 181- and 185-bp alleles of HSMS006 and the 133-, 143-, and 149-bp alleles of HSMS801 were found more often, but the 189- and 191-bp alleles of HSMS006 were much less frequent in Graves disease patients compared with the controls. An additional 174-bp allele of the HSMS602 marker, absent in healthy subjects, was found in Graves disease patients.     

Regulation of expression of mouseC4 andSlp genes by non-H-2-linked genes

C4 (the fourth complement component) and Slp (sexlimited protein) are two homologous plasma proteins encoded by genes in theS-region of theH-2 gene complex. We studied the genetic factors influencing the plasma levels of these proteins and their mRNA levels in liver. Considerable differences in both protein and mRNA levels were found between mouse strains carrying the sameS-region allele on different genetic backgrounds, indicating a pretranslational effect of non-H-2-linked genes on the expression of the twoS-region genes. The expression of Slp is androgen-dependent in the strains tested. However, testosterone treatment cannot increase the low levels of Slp caused by non-H-2-linked regulatory genes. In mice with Slp-negativeS-region alleles we found liver mRNA hybridizing with Slp-specific oligonucleotides, indicating expression of theSlp gene in Slp-negative strains. Our data demonstrate the complexity of the regulation of theC4 andSlp genes and pave the way for the analysis of the regulatory factors involved.     

Structural and evolutionary comparisons of four alleles of the mouse immunoglobulin kappa chain gene,Igk-VSer

The mouseIgK-VSer gene encodes an immunoglobulin light chain variable region which gives rise to two phenotypic polymorphisms of mouse chains. The nucleotide sequences of coding and flanking regions of theIgk-VSer ^c andIgk-VSer ^d alleles found in recently inbred strains of wild mice are compared with those of theIgk-VSer ^a andIgk-VSer ^b alleles described previously. Results suggest that the gene is evolving randomly and that framework 2 and complentarity determining region 2 are preserved, presumably for overall light chain structure. Results indicate that all four allels have an octamer motif upstream of the gene which should be functional and allow prediction of whether or not the product of the germ line gene will be detectable as either the I_B-peptide or Ef1^a phenotypic polymorphism. Southern hybridization of genomic DNA using as probe a 1-kbXba I-Xba I fragment located approximately 4 kb upstream of the BALB/cIgk-VSer ^b coding region demonstrated the presence of homologous DNA in mice bearing theIgk-VSer ^a allele and absence from mice bearing theIgk-VSer ^c andIgk-VSer ^d alleles. Nucleotide sequence comparison of BALB/c and SK/CamRk (Igk-VSer ^d ) DNA in this region demonstrated that BALB/c contained an insertion 2.4 kb in length which was absent from SK/CamRk. Both strains contain DNA homologous to the reverse complement of the mouse Bam5 repetitive element at the point of the insertion, with BALB/c containing approximately 70 nucleotides more of the element than SK/CamRk. Surprisingly, the strains containing DNA related to theXba I-Xba I probe are not those determined to be the most similar by nucleotide sequence comparisons and by the Phylogenetic Analysis Using Parsimony program. The evolutionary relationship of the alleles and a possible basis for the inconsistency presented by theXba I-Xba I fragment-related DNA are discussed.     

Mapping of the SLA complex class III region by pulsed field gel electrophoresis

The fine order of genes in the class III region of the swine major histocompatibility complex (MHC), the SLA complex, was examined by pulsed field gel electrophoresis (PFGE) and Southern blot analysis. Four genes, C2, HSP70, TNF, and CYP21, were analyzed. The CYP21, C2, and HSP70 genes were all located within a 200-kb NotI fragment. The C2, HSP70, and TNF genes cohybridized to a 420-kb SalI fragment. The TNF gene is linked to the class I region by a 390-kb NotI fragment. Combined with a previous study from our lab, the order of genes in the SLA complex is class II-class III [(CYP21/C4)-(Bf/C2/HSP70)-TNF]-class I. The size of the class III region from CYP21 to TNF is estimated to be 500 kb. This size and the order of the genes in the swine class III region are similar to those of human, mouse, goat, and rabbit, which confirms the high conservation of class III gene organization across species.     

Murine cytotoxic T-cell response to alphavirus is associated mainly withH- 2D k

A secondary in vitro response to alphaviruses Bebaru, Sindbis, and Semliki Forest is described. Optimum response appears at day 5–6 of culture. The cells responsible for lytic activity are nonadherent, -positive, Ig^–, and mainly Ly-2.1 positive. Out of five haplotypes tested (H- 2 ^d ,H- 2 ^b ,H- 2 ^s ,H- 2 ^q , andH- 2 ^k ) onlyH- 2 ^k was a responder. Genetic mapping of the response located it solely in theD region of theH- 2 complex. The other four haplotypes responded with a high antiself activity after a second stimulation with viruses. This antiself response also maps in theD region of theH- 2 complex. No complementation was observed in F₁ hybrids between responder and nonresponder strains.     

Isolation of virus-like (VL30) elements from theQ10 andD regions of the major histocompatibility complex

Previous studies from our laboratory have described two endogenous provirus-like sequences in a series of cosmids spanning theTL region of the major histocompatibility complex (MHC) of normal C57BL/10 mice. At least one of these viruses shares similarities withVL30 elements. To determine if additionalVL30-like retroviral elements are integrated in the MHC, we constructed a cosmid library using DNA from a radiation leukemia virus (RadLV)-transformed cell line derived from C57BL/6 mice. The library was first screened using theH-2III (5) probe, which detects Class I genes of theH-2 complex. In the primary screening 163H-2III positives were isolated. TheH-2III-positive isolates were then hybridized with an AKR-derived virus probe,EcoB/S, which contains sequences from both thepol and theenv genes of the virus. Nine virus-positive isolates were detected. Localization of these cosmid isolates containing viral sequences within theH-2 complex was done utilizing low-copy probes and confirmed using previously mapped cosmid isolates from other laboratories. We report here the isolation and characterization ofVL30-like elements from theQa andD regions of theMHC of several inbred mouse strains.     

Translational control inE. coli: The case of threonyl-tRNA synthetase

Genetic studies have shown that expression of theE. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5 end of the mRNA and between 10 and 50bp upstream of the translational initiation codon in thethrS gene, is directly involved in that control. The conformation of anin vitro RNA fragment extending over thethrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constitutent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.     

Isolation of four HSP70 genes in the pig and localization on Chromosomes 7 and 14

For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5 section, the central section, and the 3 section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4–2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.     

Polymorphism of the mouse gene for the interleukin 10 receptor alpha chain (Il10ra) and its association with the autoimmune phenotype

Several studies suggest that interleukin (IL)-10 pathway is involved in murine lupus, while no linkage of IL-10 gene polymorphism to disease susceptibility has been reported in studies with lupus-prone mice. Since IL-10 functions through the specific IL-10 receptor alpha (IL-10RA) chain and the IL-10RA gene (Il10ra) is linked to the susceptibility loci of atopic dermatitis and Crohn's disease identified using mouse models, we supposed that IL-10RA might be involved in murine lupus. By flow cytometry analysis, we found that NZW mice, one of the parental strains of lupus-prone (NZB×NZW) F1 mice, express extremely low levels of IL-10RA compared with NZB mice, the other parental strain, and the healthy BALB/c and C57BL/6 mice. Sequence analyses of Il10ra cDNA of NZW mice showed multiple nucleotide mutations compared with that of NZB and C57BL/6 strains, some of which would result in amino acid substitutions in the IL-10RA protein. Lupus-prone MRL mice shared the same polymorphism with NZW. Analyses using (NZB×NZW) F1×NZB backcross mice showed that high serum levels of IgG antichromatin antibodies were regulated by a combinatorial effect of the NZW Il10ra allele and a heterozygous genotype for Tnfa microsatellite locus. Our data suggest that the polymorphic NZW-type Il10ra may be involved in the pathologic production of antichromatin antibodies and, if so, may contribute in part to the development of systemic lupus erythematosus as one susceptibility allele. The Il10ra polymorphism data reported in this paper have been submitted to the Mouse Genome Informatics database and have been assigned the accession number MGI: 3528086.     

The expression ofI-region gene products on lymphocytes

Mixed lymphocyte reaction (MLR) stimulation by purified T and B lymphocytes and thymocytes was studied. The MLR gene products involved were localized to theH-2 complex by the use of congenic mice differing atH-2, and to loci within theH-2 complex through the use of congenic mice bearing recombinant chromosome 17. Stimulation by T cells was investigated in detail. The role of small amounts of contaminating B lymphocytes, and that of backstimulation, was found to be of minor importance. T cells and thymocytes stimulated as well as or better than B cells in combinations differing in theI, S, and possibly parts of theD end, thus suggesting that these genetic regions control cell-surface products expressed on both T and B lymphocyte populations.Abbreviations used in this paper are MLR mixed lymphocyte reaction - GVHR graft-versus-host reaction - CML cell-mediated lympholysis - Thy-1 the gene for the T-cell antigens, synonymous with - Thy-1.1 synonym for ^AKR - Thy-1.2 synonym for ^C3H - MHC major histocompatibility complex - Ir genes immune response genes linked to the MHC - LPS E. coli 055.35 lipopolysaccharide For the genetic nomenclature of theH-2 complex (H-2K, H-2D, I, S, D regions,Ia, etc.) see Kleinet al. 1974, and Shreffleret al. 1974.     

Localization of the upstream regulatory sites of yeast iso2-cytochrome c gene

Summary In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5 flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E. coli beta-galactosidase lacZ gene. When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c. The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1 ⁺ cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol.It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element. This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.     

A gal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cya delta) background.

Summary Strains ofEscherichia coli K12 that contain a deletion of the adenyl cyclase gene (cya), required for the synthesis of cyclic adenosine-3; 5 monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designatedgalP20) is linked to thegal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage . Studies withgalP20 cya strains as well asgal (deletions of thegal operon)cya strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function missing from both thegal strains and thegalP20 strain.     

Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced

The collectin surfactant protein-D (SP-D) plays a significant role in innate immunity. Epidemiological studies described associations between single nucleotide polymorphisms (SNPs) of the human gene coding surfactant protein-D (SFTPD) and infectious pulmonary diseases. Studies on twins indicated very strong genetic dependence for serum levels of SP-D. The aim of this study was to determine the genetic influence of sequence variations within the SFTPD gene on the constitutional serum SP-D levels. We sequenced the 5 untranslated region (5UTR), the coding region and the 3 region of the SFTPD gene of 32 randomly selected blood donors. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German blood donors. Serum SP-D levels were analysed by ELISA, and the association of SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was determined. One single SFTPD haplotype (allele frequency 13.53%) revealed a negative association with serum SP-D levels (P<0.0001). This was confirmed in a second prospectively collected group of blood donors (n=160, P=0.0034). The discovery of a frequent negative variant of the SFTPD gene provides a basis for genetic analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.     

Minimal enhancer elements of the leghemoglobinlba andlbc3 gene promoters fromGlycine max L. have different properties

The characteristics of the soybean leghemoglobinlba gene promoter were analyzed and important promoter elements from thelba andlbc3 promoters were compared using transgenicLotus corniculatus plants. A 5 deletion analysis of thelba promoter delimited twocis-acting elements controlling expression: a distal positive element (–1254, –884) required for expression and a proximal element (–285, –60) essential for full-level activity. In contrast to the corresponding region of thelbc3 promoter, thelba proximal element is unable to control expression from the heterologous CaMV 35S enhancer. The upstream positive element of thelba gene contains a position- and orientation-independent enhancer between positions (–1091, –788). The sequence of this enhancer region is conserved in thelbc3 gene upstream (–1333, –1132) of the previously assigned strong positive element (SPE; –1090, –947). The present analysis revealed some of the properties of this extendedlbc3 SPE element. The extended element (–1364, –947) functions in both orientations from 5 locations whereas the SPE2 subcomponent (–1364, –1154) containing the conserved sequence is only active in the correct orientation. Removal of the SPE2 by internal deletion demonstrates that the SPE2 subcomponent is indispensable for the activity of thelbc3 upstream positive element. These results indicate that the upstream positive elements of thelba andlbc3 genes possess different properties although their conserved minimal enhancer sequence has similar function. This may reflect the differential expression of the twolb genes ofGlycine max L.     

A pentanucleotide tandem duplication polymorphism in the 3′ untranslated region of the HLA-linked heat-shock protein 70-2 (HSP70-2) gene

A novel two-allele pentanucleotide tandem duplication polymorphism is described within the 3 untranslated region of the HLA-linked HSP70-2 gene, for which a PstI polymorphism is known. All four haplotype combinations were found.     

Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco

The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identifycis-acting regulatory sequences involved in sugar response using transgenic tobacco plants. The 5 control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium. This wound response was further enhanced by sugar. Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity. Deletion of the sequence between-892 and-573 abolished the wound response but not the sugar response. An additional 5 deletion to-453 removed the sugar inducibility. Locations of thecis-acting regulatory elements were further elucidated by 3 deletion analysis. Deletion of the downstream region from-520 did not affect the wound of sugar response of the promoter. However, 3 deletion mutant-574 was unable to respond to sugar but did respond weakly to wounding. Further deletion to-624 abolished both responses. Therefore, it can be concluded that a wound response element is located in between-624 and-574 and that the response is further enhanced by a sugar response element located in the sequence between-573 and-520.