MHC class I genes of the channel catfish: sequence analysis and expression

 Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β₂-microglobulin (β₂m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 M _r glycoprotein (presumably class I α) was associated with β₂m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β₂m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts. Received: 25 March 1998 / Revised: 28 July 1998     

Isolation and mapping of the rabbit DM genes

 Proper peptide presentation by major histocompatibility complex (MHC)-encoded class II antigens is dependent on the products of the MHC DM loci. We identified the rabbit orthologues (RLA-DMA and -DMB) of human HLA-DMA and -DMB and found that they have 76.9% and 78.8% identity with HLA-DMA and -DMB, respectively. Like classical class II MHC genes, RLA-DM genes are more closely related to human HLA-DM genes than to mouse H2-DM. Among the DM family, there is a high degree of variability at the amino terminus of the DMa chains, and length variability in the cytoplasmic tails of both DMα and DMβ. The rabbit DM genes are coexpressed with class II genes in lymphoid tissues, as are the DM genes of other mammals. The RLA-DM locus maps to the class II region of the rabbit MHC, and is flanked by the DP and DOB loci. Despite having some similarities to class II genes of bony fishes, the DM family represents a separate branch of the MHC class II family. Received: 30 May 1998 / Revised: 28 July 1998     

Allelic diversity at the Mhc-DQA locus in cotton rats (Sigmodon hispidus) and a comparison of DQA sequences within the family Muridae (Mammalia: Rodentia)

 The cotton rat (Sigmodon hispidus) is a common murid rodent of the southern United States, Mexico, and Central America. Using single-stranded conformation polymorphism analysis and DNA sequencing techniques, 11 DQA exon 2 alleles were detected among 180 S. hispidus from Caddo County, Oklahoma, USA. The alleles represent a single locus exhibiting a high level of polymorphism. Nucleotide and amino acid distance values among DQA alleles of S. hispidus were higher than those within Mus musculus and species of Rattus. Although the distribution of polymorphic amino acid residues among alleles of S. hispidus was similiar to that of Mus and Rattus, some residues of the α-helix region were more variable in S. hispidus. Comparisons of nonsynonymous and synonymous substitutions indicated a trend toward higher numbers of nonsynonymous substitutions; however, this difference was not significant statistically among S. hispidus alleles. To examine evolution of DQA alleleswithin Muridae, we performed a phylogenetic analysis that included DQA alleles from S. hispidus, Peromyscus leucopus, M. musculus, R. norvegicus, and six Australian species of Rattus. Results depicted monophyly for each genus, and this concordance between species and gene trees represents a lack of evidence for trans-species persistence of alleles among these genera. Received: 22 October 1998 / Revised: 17 March 1999     

Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex

A unicellular charophyte alga, Closterium peracerosum–strigosum–littoralecomplex (C. psl. complex), has been studied in order to obtainbasic information regarding sexual reproduction in plants. Systemsfor gene introduction and transient expression were developedfor endogenous genes using phleomycin resistance (ble) and Chlamydomonasgreen fluorescent protein (cgfp) genes as selection markers.These genes have codon usage similar to that of genes in theC. psl. complex. To drive these genes strongly into C. psl.complex cells, two native promoters of the C. psl. complex genome—CpHSP70and CpCAB1—were linked to a ble::cgfp fusion gene andintroduced into the cells by particle bombardment. Following2 d of incubation, we found 500 cells expressing GFP under thecontrol of the CpHSP70 promoter, which were identified followingheat shock treatment at 42°C, and 100 cells expressing GFPunder the control of the CpCAB1 promoter, which were observedin lit conditions. In contrast, the GFP signal was only detectedin two cells when ble::cgfp under control of the cauliflowermosaic virus 35S promoter was introduced. The ble::cgfp fusionprotein was detected in the nucleus, whereas the single cgfpprotein was detected in the cytoplasm. Our results indicatethat the newly isolated native promoters of CpHSP70 and CpCAB1are useful tools for inducing exogenous gene expression underheat shock and lit conditions, respectively. In addition, thisstrategy can be used for transient assays, such as the intracellularlocalization of unknown gene products in the C. psl. complex.     

Effects of active and passive hyperthermia on heat shock protein 70 (HSP70)

Summary. The purpose of this study was to delineate the effects of hyperthermia and physical exercise on the heat shock protein 70 (HSP70) response in circulating peripheral blood mononuclear cells (PBMCs). Six healthy, young (age: 24 ± 3 yrs), moderately trained males (VO_2max: 48.9 ± 2.7 ml · kg · min⁻¹) undertook two experimental trials in a randomised fashion in which the core temperature (T _c) was increased and then maintained at 39 °C during a 90 min bout by either active (AH) or passive (PH) means. AH involved subjects cycling at 90% of their lactate threshold in attire designed to impede heat loss mechanisms. In the PH trial, subjects were immersed up to the neck in a hot bath (40.2 ± 0.4 °C), once the critical T _c was achieved, intermittent cycling and water immersions were prescribed for the AH and PH conditions, respectively, to maintain the T _c at 39 °C. HSP70 was measured intracellularly pre, post and 4 h after trials, from circulating PBMCs using an ELISA technique. T _c reached 39 °C quicker in PH than during AH trials (PH: 21 ± 4 min vs. AH: 39 ± 6 min; P < 0.01), thereafter T _c was maintained around 39 °C (PH: 39.1 ± 0.2 °C; AH: 38.8 ± 0.3 °C; P > 0.05). AH induced a marked leukocytosis in all sub-sets (P < 0.05). PH generated significant monocytosis and granulocytosis (P < 0.05), without changes in lymphocyte counts (P > 0.05). There were no significant increases in intracellular HSP70 at 0 h (AH: Δ − 21.1 ± 44.8; PH: Δ + 12.5 ± 32.4 ng/mg TP/10³/μl PBMCs; P > 0.05) and 4 h (AH: Δ − 30.0 ± 40.1; PH: Δ + 36.3 ± 70.4 ng/mg TP/10³/μl PBMCs; P > 0.05) post active and passive heating. Peak HSP70 expressed as a fold-change from rest was also not increased by AH (1.1 ± 0.9; P > 0.05) or PH (3.2 ± 4.8; P > 0.05). There were no significant differences between the AH and PH trials at any time-point, and the HSP70 response appeared to be individual specific. These results did not allow us to delineate the effects of hyperthermia and other exercise associated stressors on the heat shock response and therefore further work is warranted. Authors’ address: Ric Lovell, Department of Sport, Health and Exercise Science, University of Hull, Hull HU6 7RX, U.K.     

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-Mog-D17Tu42-M3-M2

 A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2 ^b ) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2 ^bc ) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen –Znf173–Tctex5 – Mog–D17Tu42–D17Mit232–H2-M3–D17Leh525–H2-M2– Tel. The gene order of Znf173 – Tctex5 – Mog – D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A. Received: 25 July 1997 / Revised: 10 September 1997     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

CC-chemokine receptor 5 polymorphism and age of onset in familial multiple sclerosis

 Multiple sclerosis (MS) is a common disease of the central nervous system characterized by myelin loss and progressive neurological dysfunction. An underlying genetic susceptibility plays a clear role in the etiology of MS, likely acting in concert with an undefined environmental exposure. Full-genome screenings in multiplex MS families have identified several susceptibility regions, supporting a polygenic model for MS. Among these regions, evidence for weak linkage was observed at 3p/3cen suggesting the presence of an MS gene(s) of modest effect. Encoded here are two chemokine receptors, CCR5 and CCR2B. We examined the chromosome 3p21–24 region in 125 MS families (322 total affecteds and 200 affected sib-pairs), and performed genetic analyses of CCR5 and CCR2B loci and two nearby markers (D3S1289 and D3S1300) using both linkage- and association-based tests. No evidence of linkage to MS was observed for any of the tested markers. Affected relative-pair (SimIBD) and sib-pair analyses (ASPEX), and association testing (sib-TDT) for each locus were also not significant. However, age of onset was approximately 3 years later in patients carrying the CCR5 Δ 32 deletion (P=0.018 after controlling for gender effects). Thus, chemokine receptor expression may be associated with differential disease onset in a subset of patients, and may provide a therapeutic target to modulate inflammatory demyelination. Received: 27 October 1999 / Revised: 9 December 1999     

Linkage disequilibrium and fingerprinting in sugar beet

It has been suggested that map information for molecular markers can be used to strengthen finterprinting analyses. The success of this strategy depends on the distribution of linkage disequilibrium over the genome. Using 451 mapped AFLP markers, we investigated the occurrence of linkage disequilibrium in nine sugar beet breeding lines. A low but significant level of linkage disequilibrium was found for unlinked markers. Only for very tigthly linked (<3 cM) markers was this level substantially higher. This implies that little is gained in utilising the map position of the markers in fingerprinting applications. Received: 25 May 1999 / Accepted: 18 Oktober 1999     

Characterization and evolution of major histocompatibility complex class II genes in the aye-aye, Daubentonia madagascariensis

Major histocompatibility complex genes (Mhc-DQB and Mhc-DRB) were sequenced in seven aye-ayes (Daubentonia madagascariecsis), which is an endemic and endangered species in Madagascar. An aye-aye from a north-eastern population showed genetic relatedness to individuals of a north-western population and had a somewhat different repertoire from another north-eastern individual. These observations suggest that the extent of genetic variation in Mhc genes is not excessively small in the aye-aye in spite of recent rapid destruction of their habitat by human activities. In light of Mhc gene evolution, trans-species and allelic polymorphisms can be estimated to have been retained for more than 50 Ma (million years) based on the time scale of lemur evolution.     

Evolutionary History of MHC Class I Genes in the Mammalian Order Perissodactyla

We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. These data suggest that a diversification of MHC genes took place after the split between the Equidae and the Rhinocerotidae yet before the speciation events within the genus Equus. Received: 17 November 1998 / Accepted: 7 April 1999     

Gene-modified tumor cells as cellular vaccine

 The identification and characterization of many tumor antigens and the parallel explosion of knowledge of the cellular and molecular mechanisms of antigen recognition by the immune system have given renewed hopes that immunogenetherapy could be a promising modality to treat certain tumors. Many different novel strategies have been developed to derive genetically modified tumor cells and use them as cellular vaccines to induce useful antitumor immunity in a variety of animal tumor models. This review discusses induction of tumor immunity by injecting tumor cells that are genetically engineered to secrete various cytokines and to express major histocompatibility complex molecules and/or costimulatory molecules. While there has been a great success in inducing excellent antitumor immunity in a variety of tumor models, there are some difficulties and limitations in the application of these gene-modified tumor cells for the treatment of preexisting tumors. A number of improvements and modifications are already underway to overcome some of these problems. Received: 6 August 1996 / Accepted: 20 September 1996     

Genomic location and characterisation of MIC genes in cattle

Major histocompatibility complex (MHC) class I chain-related (MIC) genes have been previously identified and characterised in human. They encode polymorphic class I-like molecules that are stress-inducible, and constitute one of the ligands of the activating natural killer cell receptor NKG2D. We have identified three MIC genes within the cattle genome, located close to three non-classical MHC class I genes. The genomic position relative to other genes is very similar to the arrangement reported in the pig MHC region. Analysis of MIC cDNA sequences derived from a range of cattle cell lines suggest there may be four MIC genes in total. We have investigated the presence of the genes in distinct and well-defined MHC haplotypes, and show that one gene is consistently present, while configuration of the other three genes appears variable.     

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class II genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.The sequence data reported are available in the GenBank database under the accession numbers AY257165 (YFVw*15), AY257166 (YFVw*16), AY257167 (YFVIw*15), AY257168 (YFVIw*17), AY257169 (YFw*16), and AY257170 (YFw*17)