HLA class I and H ferritin gene polymorphisms in normal subjects and patients with haemochromatosis

Summary The gene for idiopathic haemochromatosis is located on the short arm of chromosome 6 within 1 cM of the HLA-A locus. In this region there are many HLA class I genes, and there may also be a gene for the H subunit of ferritin. Both HLA class I and H ferritin genes are therefore candidates for the abnormal gene in idiopathic haemochromatosis. In 15 unrelated patients the frequency of HLA-A3 was 80% compared with 24% for 600 unrelated individuals from South Wales. The most common haplotype involved is probably HLA-A3, B7. DNA was prepared from leucocytes from 12 of these patients and from 85 normal subjects. After digestion with Taq1, electrophoresis, and Southern blotting, class I sequences were detected by hybridisation to an HLA class I probe (pHLA-A). Of the 34 restriction fragments detected, 22 were polymorphic. Particular fragments correlated with the presence of HLA-A antigens A1, 2, 3, 10, 11, w19, and 28, but there was little correlation with B antigens. Restriction fragment patterns specific for haemochromatosis were not found with TaqI or during less extensive studies with other restriction enzymes. No differences in restriction fragment patterns were found between four patients and four normal subjects apparently homozygous for HLA-A3 and B7. Examination of Southern blotting patterns for genomic DNA from patients and normal subjects with a panel of 12 restriction enzymes and a probe for the H ferritin gene (pDBR-2) revealed no polymorphisms associated with either idiopathic haemochromatosis or particular HLA phenotypes. These studies provide no support for either HLA class I genes or the H ferritin gene as candidates for the haemochromatosis gene.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

The chromosome region containing the highly polymorphic HLA class I genes displays limited large scale variability in the human population

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.     

Physical and genetic mapping of the telomeric major histocompatibility complex region in man and relevance to the primary hemochromatosis gene (HFE).

We performed pulsed-field gel electrophoresis (PFGE) on genomic DNA from a radiation hybrid (RH) cell line and constructed a high-resolution physical map of the major histocompatibility complex class I region in 6p21.3, where the gene for primary hemochromatosis (HFE) is believed to be located. Due to the intact microsegment of hemizygous human genomic DNA preserved in the RH cell line, simplified and distinct restriction fragment banding patterns were generated. Using the RH cell line, we were able to extend the physical map of the HLA class I region to about 3000 kb, order the known HLA class I genes from centromere to telomere: HLA-B, -C, -E, (-A, -H, -G), and -F, and orient the HLA-F gene along the chromosome. The proximity of HLA-F to HLA-A was confirmed by linkage and linkage disequilibrium analysis. This study shows that RH cell lines can be useful for constructing long-range physical maps in specific regions of the human genome with PFGE. Physical and genetic mapping studies of this region are consistent with a localization of the HFE gene proximal or distal to HLA-A.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

HLA-DR4-associated haplotypes are genotypically diverse within HLA

Biochemical diversity among products of class II HLA genes has been observed in individuals who appear to be HLA-D and DR-identical by cellular and serologic typing. We used techniques of restriction enzyme fragment analysis by Southern blotting to analyze this diversity at the level of cellular DNA. A panel of 17 HLA-DR4 homozygous cell lines (HCL) were investigated by using cDNA probes homologous to DQ beta, DQ alpha, and DR beta genes. Each probe was hybridized to cellular DNA digested with a series of different restriction endonucleases. Polymorphisms were observed with the use of the enzymes Pst I, Hind III, and Bam HI: Hybridization of cellular DNA digested with Hind III and Pst I with the DQ beta probe revealed specific polymorphisms, as did hybridization of the Pst I digest with the DQ alpha cDNA probe and the Bam HI digest with the DR beta probe. The observed differences fall into two categories: first, considerable diversity was seen between HLA-DR4 HCL that represent different HLA-D-defined haplotypes; second, diversity was also observed among HCL of the same DR4-associated HLA-D cluster. In contrast to the DQ cDNA probes, hybridization with the DR beta probe revealed relatively limited polymorphism by using a panel of different restriction endonucleases. Thus, although there is a general pattern of polymorphic restriction enzyme fragments homologous to DQ probes within an HLA-D cluster, the pattern seen for any particular cell line was not sufficiently distinct to assign an HLA-D or DR specificity.     

SMX-1 virus-induced inhibition of ecotropic and recombinant proviral sequence amplification in thymuses of AKR mice

SMX-1 virus delays the appearance of spontaneous thymomas in AKR mice which have been inoculated as young adults by the intrathymic route. Analyses of high-molecular-weight thymus DNAs from SMX-1 virus-inoculated AKR mice indicated the absence of 3' recombinant proviral-cellular DNA junction fragments generated by EcoRI and PvuII digestion. An average of five recombinant proviral fragments were detected in DNAs from spontaneous thymomas that developed in medium-injected control mice. Preleukemic mice that amplify murine leukemia virus-related antigens on their thymocyte surface contained unintegrated proviruses in their thymus DNAs, and 2.3-kilobase EcoRI and 2.1-kilobase PvuII recombinant DNA fragments were detected.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.     

Transfer of cloned human class I major histocompatibility complex genes into HLA mutant human lymphoblastoid cells.

Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.     

First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination

Summary The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis.     

Association of restriction fragment length polymorphisms of swine leucocyte antigen class I genes with production traits of Duroc and Hampshire boars

Restriction fragment length polymorphism analyses of swine leucocyte antigen (SLA) class I genes were performed on 70 Duroc and 38 Hampshire boars from the 1986-87 national performance tests of each breed in the USA. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA, isolated from white blood cells, using PvuII endonuclease and a swine major histocompatibility complex (MHC) class I probe. Durocs had an average of 11 restriction fragments, with the most common being in 63% of the boars and the least common appearing in only one boar. Hampshire boars had an average of 12 restriction fragments, with the most common appearing in 73% of the boars and the least common appearing in only one boar. Least squares procedures and stepwise regression methods were used to examine the association between DNA restriction fragments and the selection index (INDEX), average daily gain (ADG), average backfat thickness (BF), loin muscle area (LEA), and age at 104 kg (DAY104). In the Duroc breed one DNA restriction fragment was associated with decreased INDEX (P less than 0.05) and decreased ADG (P less than 0.05) whereas two other fragments were associated with increased BF (P less than 0.05). In the Hampshire breed two restriction fragments were associated with an increase in INDEX (P less than 0.05). Cluster analyses were used to group pigs of each breed on the basis of similar RFLP patterns. One cluster group in the Duroc breed was associated with lower average INDEX values (P less than 0.05), greater average DAY104 (P less than 0.05), and a larger mean LEA (P less than 0.05). In the Hampshire breed one cluster group was associated with lower INDEX (P less than 0.05). These results suggest there may be an association between swine MHC class I genes and performance traits in swine. The use of SLA class I restriction fragments, as genetic markers, may have potential in the future for improving pig performance.     

Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica napus

Summary Chloroplast (cp) and mitochondrial (mt) compartments of normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been characterized and compared on the basis of cp and mt DNA restriction enzyme analysis and in vitro protein synthesis by isolated mitochondria. Cytoplasmic male sterility of B. napus (rape) comes from cms Raphanus sativus (radish) through intergeneric crosses.Cp DNAs isolated from N and cms lines had distinct restriction patterns with Sal I, Kpn I and Sma I enzymes. The size of the two cp DNAs measured from the restriction patterns was found to be identical and of about 95 × 10⁶ d. N and cms lines of B. napus were characterized by specific mt DNAs, as shown from Sal I, Kpn I, Pst I and Xho I cleavage patterns. The small number of well-separated restriction fragments obtained with Sal I enabled us to determine precisely mt DNA sizes. The values of 136.5 and 140.3 × 10⁶ d, obtained from restriction patterns with N and cms DNAs respectively, are smaller than any of those previously obtained from studies on other genera. With molecular hybridization experiments, it was possible to distinguish N and cms lines by the different locations of rRNA genes on the cp and mt DNAs.Two lines of B. napus are characterized by specific mt translation products formed in isolated mitochondria.     

Activation of HLA-restricted EBV-specific cytotoxic T cells does not require CD4+ (helper) T cells or exogenous cytokines

MHC-restricted, viral Ag-specific "memory" CTL are thought to play a decisive role in the defense against pathogenic viruses. However, the requirements for activating such CTL remain controversial. In particular, the role of CD4+ helper cells and their soluble products (e.g., IL-2) are uncertain. To approach these questions as they relate to EBV-specific CTL, highly purified CD8+ T cells from healthy EBV-seropositive individuals were cultured with autologous irradiated EBV-transformed B lymphoblastoid cell lines (LCL), in the presence or absence of autologous CD4+ cells or 1 to 10 U/ml purified rIL-2. The results indicate that the induction of CTL requires neither Th cells nor exogenous IL-2. The CTL generated from isolated CD8+ cells were HLA class I restricted as demonstrated by their ability to lyse targets sharing at least one HLA-A or -B Ag with the stimulating autologous LCL. Furthermore, a mAb (W6/32) to a common determinant on HLA class I Ag blocked both the generation and effector phases of killing, whereas an HLA class II directed mAb had no effect. Addition of an IL-2R-specific antibody (anti-Tac) to the culture medium blocked induction of CTL, suggesting that endogenously produced IL-2 plays an obligatory role in this system. Paraformaldehyde fixation of LCL abrogated their ability to function as stimulator cells; however, addition of 2 U/ml exogenous IL-2 to fixed LCL cultured with CD8+ cells allowed for the induction of highly specific CTL. These results indicate that EBV-specific memory CTL can be activated in the absence of CD4+ helper cells or their soluble products, but nonetheless require Ag and IL-2.     

Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency

Summary DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: (i) The late onset form is associated with a double dose of a 14 kb fragment generated by Eco RI and with a triple dose of a 3.2 kb fragment generated by TaqI in patients with HLA B14 haplotypes; (ii) The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by TaqI in patients with HLA Bw47 haplotypes. A 3.2 kb TaqI fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.     

Establishment of a physical and genetic map for bacteriophage PRD1

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.     

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.     

Presence of an expressed β-tubulin gene (TUBB) in the HLA class I region may provide the genetic basis for HLA-linked microtubule dysfunction

An expressed -tubulin gene (TUBB) has previously been localized to chromosome region 6pter-p21 in man. By using a panel of deletion mutant cell lines and radiation-reduced hybrids containing fragments of chromosome 6, the TUBB locus could be mapped to the HLA class I region at 6p21.3. A long range restriction map including TUBB and several HLA class I genes was then generated by rotating field gel electrophoresis. The results show that TUBB maps to a segment 170–370 kb telomeric of HLA-C. This location suggests that a mutation at the TUBB locus could be the cause for certain forms of HLAlinked microtubule dysfunction, including immotile cilia syndrome.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

A physical map including a new class I gene (cda12) of the human major histocompatibility complex (A2/B13 haplotype) derived from a monosomy 6 mutant cell line

To avoid interpretative problems due to restriction fragment length polymorphisms, the monosomy 6 mutant cell line BM19.7 was employed to establish a molecular map of the human major histocompatibility (HLA) complex in the A2,B13,Bw4,DRw6,DRw52,DQw1,DPw2 haplotype. Results were obtained mainly by field-inversion gel electrophoresis and Southern blotting techniques. The map extends to 4800 kb and includes the HLA complex with a length of 4200 kb. Five HTF islands could be positioned on the map. The class I region has a size of about 2000 kb and includes nonclassical HLA class I genes, some of which must be localized within 200 kb telomeric of HLA-A. A new class I gene, cda12, distinct from HLA-A, HLA-B, or HLA-C, has been localized within 50 kb from HLA-A. The class I region contains a gap of about 500 kb, just telomeric of HLA-C, in which further class I genes could not be detected. The class II region has a size of 1000 kb, which is separated from the class I region by about 1200 kb. The 5' end of the HLA-B gene is situated centromeric, giving an orientation opposite to that of the TNFA and TNFB loci. The estimated length of the HLA complex correlates well with its size determined cytogenetically using mutant cell lines with interstitial deletions.