Possible role for COMT in psychosis associated with velo-cardio-facial syndrome.
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Abnormalities, Multiple↗
Biomedical subjects
I Dunham
Publications and source records attributed to I Dunham.
Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction.
Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.
Animals↗
The development and application of automated gridding for efficient screening of yeast and bacterial ordered libraries.
An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.
Animals↗
Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.
The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.
Base Sequence↗
A panel of human chromosome 22-specific sequence tagged sites.
A panel of 29 sequence tagged sites (STSs) covering the long arm of chromosome 22 has been assembled. STS primer pairs were synthesized using available chromosome 22 sequence derived from the GenBank and EMBL DNA sequence databases, as well as published cDNA and genomic sequence, or from previously published and communicated primer pairs. Each STS was optimized for the polymerase chain reaction using a chromosome 22-only hybrid and human genomic DNA. Further STS content analysis on a panel of somatic cell hybrids that incorporated two chromosome 22 translocations resulted in the mapping of the X-box binding protein (XBP), D22S156, and transcobalamin II (TCN2) genes to 22q11-q13.1. The panel of STSs was used for the rapid determination of the STS content and thus the chromosomal DNA content of a new irradiation hybrid.
Base Sequence↗
A random STS strategy for construction of YAC contigs spanning defined chromosomal regions.
Sequence tagged sites (STSs) that were generated via Alu-element-mediated polymerase chain reaction (Alu-PCR) and mapped to human Xq26 were used to isolate and overlap yeast artificial chromosomes (YACs). By collating the results of primary pool screening, the order of STSs and YACs was postulated directly. Subsequent isolation of 11 key YACs from 75 positive pools confirmed the proposed contig. Although only a small subset of the available Alu-PCR fragments was used, the STSs were generated at sufficient density to isolate all the YACs required and to identify all except one overlap directly. The results confirmed physical linkage of HPRT to DXS86 and DXS144E. Long-range continuity was determined purely by analysis of the 11 YAC colonies and required no end-rescue. This strategy is therefore an effective approach for the construction of YAC contigs spanning discrete chromosomal regions contained within somatic cell hybrids, with minimal prior knowledge of the region.
Base Sequence↗
Molecular analysis of the human MHC class I region in hereditary haemochromatosis. A study by pulsed-field gel electrophoresis.
The unknown allele that predisposes to the development of haemochromatosis in man has been localized to the HLA class I region on the short arm of chromosome 6. We have utilized pulsed-field gel electrophoresis in conjunction with probes that map within, or in the vicinity of, this region to search for structural lesions that may further define the disease locus. Using the enzyme Mlu I, fragments that associated specifically with the HLA-A23, A31 and B8 alleles were identified. However, in members of three pedigrees affected by haemochromatosis, and in six unrelated patients with the disorder, no disease-specific differences were detected in the DNA fragments with four restriction enzymes and eight probes when compared with healthy individuals. These data suggest that the lesion responsible for hereditary haemochromatosis lies beyond the resolution of this technique and does not involve large structural deletions or extensive re-arrangements in this highly polymorphic region of the genome.
Alleles↗
Characterization of the class III region in different MHC haplotypes by pulsed-field gel electrophoresis.
The class III region of the human major histocompatibility complex (MHC) in seven HLA haplotypes has been analyzed using pulsed-field gel electrophoresis (PFGE), restriction enzymes that cut genomic DNA infrequently, and Southern blotting. In particular, extensive mapping with the enzyme Bss HII, which generates fragments in the size range 8-280 kilobases (kb), has revealed that in the haplotypes studied the DNA content of the class III region does not appear to vary other than as previously observed at the C4 and CYP21 loci.
Blotting, Southern↗
Rapid assessment of S. cerevisiae mating type by PCR.
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Base Sequence↗
Polymorphism of the human complement component C4.
The genes encoding the two C4 isotypes, C4A and C4B, lie 10 kb apart in the class III region of the human major histocompatibility complex. The two isotypes exhibit extensive structural polymorphism. Characterisation of a number of C4A and C4B alleles has established the pattern of polymorphism in C4 and this has provided a structural basis for the observed functional and serological differences between the C4 isotypes. An intriguing feature in the genetics of C4 is the unusually high frequency of null alleles forming half null C4A and C4B haplotypes. Duplication of one of the loci has also been recognised. In addition the genes can differ in size due to the presence or absence of a large intron near the 5' end of the genes. These differences in gene size and gene number can be observed directly on different haplotypes using pulsed field gel electrophoresis.
Alleles↗
Direct observation of the gene organization of the complement C4 and 21-hydroxylase loci by pulsed field gel electrophoresis.
Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies.
Cell Line↗
Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region.
Chromosome walking in the major histocompatibility complex (MHC) class III region has resulted in the isolation of 541 kb of genomic DNA in two sets of overlapping cosmid clones. These two sets encompass the 340 kb separating the C2 and tumour necrosis factor (TNF) alpha and beta genes, except for a 22 kb gap 108 kb centromeric to the TNF alpha gene. The genomic DNA inserts have been characterized for the presence of clusters of restriction sites with CpG dinucleotides in their recognition sequence. In conjunction with pulsed field gel electrophoresis the exact sites which cleave in chromosomal DNA have been established and this has suggested the presence of a number of HTF-islands. Genomic probes flanking the HTF-islands have been hybridized to Northern blots of RNA from a number of cell lines. Transcripts ranging in size from 0.6 to 6 kb corresponding to the products of 12 novel, single copy genes have been identified. In addition the human equivalent of the murine B144 gene was mapped approximately 10 kb centromeric of the TNF alpha gene. The location of so many new genes in this region raises the question as to whether they play any role in the observed HLA associations with an individual's susceptibility to develop autoimmune disease.
Base Sequence↗
An analysis of variation in the long-range genomic organization of the human major histocompatibility complex class II region by pulsed-field gel electrophoresis.
The class II region of the human major histocompatibility complex in seven common HLA haplotypes has been analyzed using pulsed-field gel electrophoresis, restriction enzymes that cut genomic DNA infrequently, and Southern blotting. This analysis has revealed that there are differences in the amount of DNA present in the DQ and DR subregions dependent on the haplotype. The class II region of the DR3 haplotype spans approximately 750 kb and has the same amount of DNA as the class II region of the DR5 and DR6 haplotypes. However, the DR2 haplotype has approximately 30 kb more DNA within the DR subregion. The DR4 haplotype has an additional approximately 110 kb of DNA within the DQ or DR subregions compared to the DR3, DR5, and DR6 haplotypes. These haplotype-specific differences could have some bearing both on the analysis of disease susceptibility and on the ability of chromosomes possessing different HLA haplotypes to recombine within the DQ/DR subregions.
Blotting, Southern↗
Human major histocompatibility complex contains genes for the major heat shock protein HSP70.
Little is known as to why a large number of human diseases are influenced by the major histocompatibility complex. In some cases, a direct involvement of the products of the polymorphic class I and class II, aas well as the less variable products of the class III, genes has been proposed. During characterization of the class III region for the presence of additional loci, we have located a duplicated locus encoding the major heat shock protein HSP70 between the complement and tumor necrosis factor genes. The HSP70 loci are 12 kilobases apart and lie 92 kilobases telomeric of the C2 gene. As HSP70 proteins have been linked with a protective role during and after cellular stress, and HSP70 analogues are often presented as antigens in bacterial and protozoal infections, this finding may have major implications with regard to the major histo-compatibility complex and associated diseases.
Amino Acid Sequence↗
Molecular mapping of the HLA-linked complement genes and the RCA linkage group.
Phenotypic genetics have established linkage of the genes encoding proteins involved in the activation of the complement component C3. C2, factor B and C4, three of the structural components of the classical and alternative pathway C3 convertases, are encoded by genes which have been mapped to the class III region of the major histocompatibility complex (MHC) on human chromosome 6. The regulatory proteins factor H, C4BP, CR1, CR2 and DAF, which are involved in the control of C3 convertase activity, are encoded by closely linked genes, termed the regulators of complement activation (RCA) linkage group, that have been mapped to human chromosome 1. cDNA clones for all these proteins have been isolated, and this has made it possible to investigate the organization and structure of the MHC class III genes and the genes in the RCA linkage group. This short review summarizes some of the main features which have emerged from recent cloning work.
Animals↗
Molecular characterization of the HLA-linked steroid 21-hydroxylase B gene from an individual with congenital adrenal hyperplasia.
21-Hydroxylase deficiency which causes congenital adrenal hyperplasia is one of the most common defects of adrenal steroidogenesis. There are two 21-hydroxylase genes in man, A and B, and these have been mapped to the HLA class III region. Only the 21-hydroxylase B gene is thought to be active. To understand the molecular basis of congenital adrenal hyperplasia in a patient with the salt-wasting form of the disease, we cloned and characterized his single 21-hydroxylase B gene. The nucleotide sequence of this gene and a 21-hydroxylase B gene from a normal individual have been determined. Comparison of the two sequences has revealed 11 nucleotide alterations, of which two are in the 5' flanking region, four are in introns, one is in the 3' untranslated region and four are in exons. Two of the differences in exons cause codon changes, with Ser-269 and Asn-494 in the normal 21-hydroxylase B gene being converted to Thr and Ser, respectively. These amino acid substitutions may give an insight into those residues necessary for 21-hydroxylase enzymatic activity. We have also confirmed that the 21-hydroxylase A gene is a pseudogene due to three deleterious mutations in the exons. In addition, comparison of the 21-hydroxylase B gene sequence with other published sequences indicates that this microsomal cytochrome P-450 may be polymorphic.
Adrenal Hyperplasia, Congenital↗
Molecular mapping of the human major histocompatibility complex by pulsed-field gel electrophoresis.
Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC). We have isolated approximately equal to 230 kilobases (kb) of genomic DNA in overlapping cosmid clones covering the genes for the second and fourth components of complement (C2 and C4, respectively), factor B, and steroid 21-hydroxylase, and approximately equal to 82 kb of genomic DNA surrounding the genes for the tumor necrosis factors alpha and beta. Single-copy hybridization probes isolated from these cosmid clusters and probes for the known MHC gene loci were hybridized to Southern blots of genomic DNA that had been digested with infrequently cutting restriction endonucleases and separated on pulsed-field gels. The data obtained allowed the construction of a long-range genomic restriction map and indicated that the MHC spans 3800 kb. This map orients the MHC class III gene cluster with respect to the DR subregion; the C2 gene is on the telomeric side of the 21-hydroxylase B gene. In addition we have defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC. Genes for the alpha chain of DR and 21-hydroxylase B are separated by at least 300 kb, while the distance between the genes for C2 and tumor necrosis factor alpha is 390 kb. The HLA-B locus lies approximately equal to 250 kb on the telomeric side of the tumor necrosis factor genes.
Chromosome Mapping↗