The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntington's disease is located within the telomere YAC clone.


Pig standard bivariate flow karyotype and peak assignment for chromosomes X, Y, 3, and 7. A standard pig flow karyotype (2N = 38 chromosomes) was defined by standardization of several flow karyotypes obtained from stimulated peripheral blood lymphocytes of normal male and female pigs. Depending on the animals under study, the flow analysis of their chromosome suspensions gave rise to bivariate flow karyotypes comprising from 15 to 17 peaks, of which 11 to 15 represented single chromosomes. The results were used to propose a peak nomenclature. In addition, a male miniature pig lymphoblastoid cell line was characterized by flow cytogenetics. A very high-resolution flow karyotype, in which all peaks but one superimposed on those of the standard karyotype, was obtained. Peaks were assigned for chromosomes X and Y. Analysis of flow karyotypes obtained from translocated t(3,7)(p1.3;q2.1) pigs combined with polymerase chain reaction (PCR) studies of major histocompatibility complex (MHC)-linked sequences on flow-sorted chromosomes allowed identification of peaks 3 and 7 of normal pig chromosomes and of the derivative chromosomes associated with the t(3,7)(p1.3;q2.1) translocation.


The defensin-related murine CRS1C gene: expression in Paneth cells and linkage to Defcr, the cryptdin locus. The site of defensin-related CRS1C gene expression in mouse small bowel and the chromosomal location of the CRS1C locus, Defcr-rs1, have been determined. CRS1C (cryptdin-related sequence 1C) mRNA is an abundant small intestinal sequence that exhibits extensive similarity to the prepro-coding regions of defensin mRNAs yet does not encode a defensin (A. J. Ouellette and J. C. Lualdi, 1990, J. Biol. Chem. 265: 9831-9837). Using sequence-specific probes, CRS1C mRNA was detected in Paneth cells at the base of intestinal crypts by in situ hybridization. Southern blot analysis of genomic DNAs from inbred and recombinant inbred (RI) mouse strains, also conducted with probes specific for CRS1C, showed that the CRS1C locus maps to the proximal region of Chromosome 8. In 62 RI strains, no discordancies were found between Defcr-rs1 and Defcr, the cryptdin gene. Thus, both the Defcr-rs1 and the Defcr genes are expressed in Paneth cells and both are genetically inseparable within 1.58 cM on Chromosome 8. These studies identify a second defensin-related Paneth cell gene in mice.


Identification of three new genes and estimation of the size of the carcinoembryonic antigen family. Using carcinoembryonic antigen (CEA) subgroup-specific degenerate PCR primers, we have identified three new CEA gene family member L/N exons (CGM9, CGM10, and CGM11) and all previously reported L/N exons of the CEA subgroup (CEA, BGP, NCA, CGM1, CGM2, CGM6, CGM7, and CGM8). This suggests that the CEA subgroup contains 11 genes. CGM9, CGM10, and CGM11 seem to be pseudogenes. A deletion of an asparagine in CGM9 results in loss of a glycosylation site, which is conserved throughout the CEA gene family. We have previously suggested the number of genes in the pregnancy-specific glycoprotein (PSG) subgroup to be 11, which together with this study indicates that the CEA gene family contains 22 genes in all. Parsimony analysis of the CEA subgroup interrelationships suggests that CGM7 occupies the most primitive position within the CEA subgroup, being a sister group to the rest. CEA, BGP, NCA, and CGM1 form a fairly well-supported group within the CEA subgroup.


Detection of a polymorphism within the pepsinogen C gene with PCR: construction of a linkage map around PGC from 6p11-6p21.3. An insertion/deletion polymorphism between exons 7 and 8 of the pepsinogen C gene (PGC), previously detectable with Southern analysis, was formatted for detection with PCR. Alleles were rapidly typed by UV irradiation of ethidium bromide-stained agarose gels. Whereas Southern analysis revealed two alleles, the smaller fragments generated with PCR allowed the resolution of three alleles that were previously scored as a single allele and increased the heterozygosity of the system from 0.20 to 0.53. After a set of reference families was genotyped with the PCR-based polymorphism, a linkage map around the PGC gene on chromosome 6 was constructed. This included the HLA cluster and the highly informative D6S223 locus. PGC lies 22 cM proximal to HLA-DPB and between D6S5 and D6S4 at distances of 4.5 and 13.1 cM, respectively.


Feline arylsulfatase B (ARSB): isolation and expression of the cDNA, comparison with human ARSB, and gene localization to feline chromosome A1. Arylsulfatase B (ARSB) is the lysosomal enzyme that catalyzes the hydrolysis of 4-sulfate groups from N-acetylgalactosamine 4-sulfate moieties on the glycosaminoglycans, dermatan sulfate and chondroitin sulfate A. In man, a deficiency of this enzymatic activity causes the lysosomal storage disorder, Maroteaux-Lamy disease (mucopolysaccharidosis Type VI; MPS VI). MPS VI in Siamese cats also has been described, and the comparative pathologic and biochemical abnormalities of the human and feline disorders have been well characterized. The present study describes the isolation and expression of cDNAs encoding feline ARSB and the assignment of the feline ARSB gene to feline chromosome A1. The full-length feline ARSB cDNA sequence is 1939 bp, including 3 and 328 bp of 5' and 3' untranslated sequences, respectively, and a 1608-bp open reading frame encoding 535 amino acids. The predicted human and feline ARSB proteins are 91% identical and 94% similar. However, despite this high homology, the predicted feline ARSB polypeptide has nine cysteine residues, while the human enzyme has eight. The presence of the extra cysteine residue at position 451 in the feline enzyme may explain why feline ARSB is a homodimer and the human enzyme is a monomer. To facilitate comparative structure/function studies of the human and feline enzymes and to initiate somatic gene therapy trials in the MPS VI cats, a full-length feline ARSB cDNA was reconstructed from a 1440-bp partial cDNA and an ARSB fragment amplified from feline first-strand cDNA by the polymerase chain reaction. The functional integrity of this cDNA was demonstrated by transient expression in human embryonic kidney cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Detailed mapping around the breakpoint of (3;8) translocation in familial renal cell carcinoma and FRA3B. As a critical step toward cloning the breakpoint of the (3;8) translocation observed in familial renal cell carcinoma and a common fragile site on chromosome 3p (FRA3B), we have characterized the 3p14 region containing the breakpoint and FRA3B by fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), and genetic linkage analysis. Of 23 cosmids mapped by FISH, 14 cosmids were distal to the breakpoint of t(3;8) and 9 were proximal. Analyses of FRA3B by FISH were identical to those for the (3;8) breakpoint and indicated that the breakpoint of t(3;8) occurred at or very close to the fragile site. We have also constructed a genetic linkage map and a preliminary long-range restriction map using PFGE of the 3p14 region. The linkage results were consistent with the physical data. The combined results of FISH, PFGE, and linkage analysis establish a basis for further experiments to clone the (3;8) breakpoint and FRA3B.


A somatic cell hybrid panel for regional mapping of human chromosome 18. We have produced and characterized a panel of seven somatic cell hybrids defining eight potential intervals on human chromosome 18. Using 24 markers that have previously been assigned to this chromosome, we have placed markers into six of these regions. The gene for alpha 2-plasmin inhibitor (PLI) and the anonymous DNA segment locus D18S23 are excluded from this chromosome.


Length and sequence variation in the apolipoprotein B intron 20 Alu repeat. We have developed a single-stranded conformation polymorphism (SSCP) protocol for typing both sequence and length variations in an Alu element located in intron 20 of the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction (PCR), we simultaneously amplified and isotopically labeled the apo B intron 20 Alu. The Alu tail, which is composed of two arrays of variable numbers of tandem repeats, (TTTX)y (X = A or G) and (T)z, was separated from the rest of the PCR product by restriction enzyme digestion with PstI. Length variation in the Alu tail (IN20-REP) was thus separated from sequence variation in the Alu body (IN20-SEQ), rendering the SSCP patterns both eaiser to interpret and more informative. In a sample of 242 unrelated individuals from Nancy, France, we observed 11 SSCP alleles at the IN20-SEQ locus that differed only in sequence. At the IN20-REP locus, we observed 7 alleles that differed in both sequence and length. All alleles at both loci were subcloned and sequenced. One additional allele that did not undergo a detectable mobility shift in SSCP gels was uncovered at each locus during sequencing of the SSCP alleles. The additional IN20-SEQ allele was typed by restriction enzyme digestion. Although the number of IN20-SEQ and IN20-REP alleles was large, most were uncommon; the three most common alleles at each locus represented more than 94% of those sampled. We also typed the children of the 242 unrelated French individuals, enabling verification of the Mendelian segregation of the two loci and construction of haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)


The human Pi class glutathione transferase sequence at 12q13-q14 is a reverse-transcribed pseudogene. A previous in situ hybridization study with a Pi class glutathione S-transferase cDNA probe revealed the presence of hybridizing sequences on the long arms of chromosomes 11 and 12. Since the GSTP1 gene is known to be on chromosome 11 and since it is thought that chromosomes 11 and 12 arose from an ancient tetraploidization event, it was of interest to determine if the gene on chromosome 12 encoded a closely related Pi class glutathione S-transferase isoenzyme. This gene has now been cloned and sequenced. The results are surprising and indicate that the gene is a partial reverse-transcribed pseudogene that has been inserted into the genome at 12q by chance and has not resulted from the prior tetraploidization of the human genome.


The human galactose-1-phosphate uridyltransferase gene. Classical galactosemia is an inborn error of metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). Standard treatment with dietary galactose restriction will reverse the potentially lethal symptoms of the disease that are manifest in the newborn period. However, the long-term prognosis for these patients is variable. As a first step toward investigating the molecular basis for phenotypic variation in galactosemia, we have cloned and sequenced the entire gene for human galactose-1-phosphate uridyltransferase. This gene is organized into 11 exons spanning 4 kb. In exons 6, 9, and a portion of 10, there is a high degree of amino acid sequence conservation among Escherichia coli, yeast, mouse, and human. We have identified a number of nucleotide changes in the GALT genes of galactosemic patients that alter conserved amino acids. The most common of these is an A to G transition at nucleotide position 1470, converting a glutamine to an arginine at amino acid codon position 188 (Q188R).(ABSTRACT TRUNCATED AT 250 WORDS)


Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21. A library of yeast artificial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Double screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 +/- 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in situ hybridization, 17 mapped to chromosome 21.


Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 gene encoding human Ca(2+)-pumping ATPase isoform PMCA2. The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) is responsible for maintaining calcium homeostasis in eukaryotic cells. The Ca(2+)-ATPase is a family of pumps that are encoded by at least four genes. A cDNA for the human version of Ca(2+)-ATPase isoform PMCA2 was isolated and characterized. Comparison of the human and rat cDNA sequences showed that they were 95% homologous in the coding domain, and this homology was reflected in the deduced protein sequence where greater than 98% homology between the human and rat sequences was found. The amino acid differences that were found were almost all conservative. The PMCA2 cDNA was used to probe Southern blots of human-rodent somatic cell hybrid DNAs; the results indicated that the human PMCA2 gene was located on chromosome 3.


The gene for the cell adhesion molecule M-cadherin maps to mouse chromosome 8 and human chromosome 16q24.1-qter and is near the E-cadherin (uvomorulin) locus in both species. A mouse myotube-derived cDNA encoding the Ca(2+)-dependent cell adhesion molecule M-cadherin was used to study the segregation of the corresponding gene Cdh3 in a mouse interspecific backcross. Cdh3 was found to be unlinked to the N-cadherin gene but linked to the E-cadherin (uvomorulin) locus on chromosome 8 in a region of conserved synteny with human chromosome 16q. The gene order cen-Junb-Um-Tat-(Cdh3/Aprt) was determined. The human homologue CDH3 was mapped to chromosome 16q24.1-qter by analyzing human/mouse somatic cell hybrids.


Neural retina-specific leucine zipper gene NRL (D14S46E) maps to human chromosome 14q11.1-q11.2. The product of a neural retina-specific gene, NRL, belongs to the "leucine zipper" family of DNA-binding proteins and has a strong similarity to the v-maf oncogene product. The NRL gene maps to human chromosome 14 by Southern blot analysis of genomic DNA from a human-rodent somatic cell hybrid panel. In situ hybridization to metaphase chromosomes has further sublocalized the gene to the region 14q11.1-q11.2. D14S46E has now been assigned to the NRL gene. Because of its specific pattern of expression, NRL is a candidate gene for retinal diseases.


Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21. Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid beta protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.


Genetic mapping of the integrin alpha 1 gene (Vla1) to mouse chromosome 13. The integrin alpha 1 chain (Vla1) associates with the beta 1 chain to form a heterodimer that functions as a dual laminin/collagen receptor in neural cells and hematopoietic cells. We have used an interspecies backcross gene-mapping technique to map the Vla1 gene to the distal end of chromosome 13 in the mouse genome. The Vla1 locus is located 3.5 cM distal to Ctla-3 and 7.8 cM distal to Htrla. We have further characterized this locus in recombinant inbred (RI) mice by examining the strain distribution patterns of nine genomic DNA restriction fragment length variants detected with alpha 1 cDNA probes. The RI gene mapping did not show linkage to previously mapped genes or mutants in the AXB, BXA, or AKXD RI sets and therefore defines a new genetic marker for the distal end of chromosome 13 in these RI sets.


Linkage mapping of the AML1 gene on human chromosome 21 using a DNA polymorphism in the 3' untranslated region. We have detected a polymorphism in the 3' untranslated region of the AML1 gene, which is located at the breakpoint on chromosome 21 in the t(8;21)(q22;q22.3) translocation often associated with patients with acute myeloid leukemia. Informative CEPH families were genotyped for this polymorphism and used to localize the gene on the linkage map of human chromosome 21. The AML1 gene is located between the markers D21S216 and D21S211, in chromosomal band 21q22.3.


The gene for X-linked hydrocephalus maps to Xq28, distal to DXS52. We report the study of five independent X-linked hydrocephalus (HSAS1) families with polymorphic DNA markers of the Xq28 region. A total of 58 individuals, including 7 living affected males and 22 obligate carriers, have been studied. Maximum lod score was 7.21 at theta = 2.40% for DXS52 (St14-1). A single recombination event was observed between this marker and the HSAS1 locus. Other markers studied were DXS296 (Z = 2.02 at theta = 2.5%), DXS304 (Z = 4.37 at theta = 7.8%), DXS74 (Z = 3.50 at theta = 0%), DXS15 (Z = 1.96 at theta = 5.7%), DXS134 (Z = 3.31 at theta = 0%), and F8C (Z = 5.79 at theta = 0%). These data confirm the localization of the HSAS1 gene to Xq28 and provide evidence for genetic homogeneity of this syndrome. In addition, examination of two obligate recombinant meioses along with multipoint linkage analysis supports the distal localization of the HSAS1 locus with respect to the DXS52 cluster. These observations are of potential interest for future studies aimed at HSAS1 gene characterization.


Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction. alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.


Chromlook: an interactive program for error detection and mapping in reference linkage data. Preliminary genetic linkage maps of every human chromosome have been generated over the past few years, and efforts to extend and refine these maps are under way. However, fine-resolution mapping is tedious and difficult because the inevitable errors in the data confound estimates of both the placement of loci and the distances between them. Fortunately, in most cases these errors result in observed recombinants where no true recombinant has occurred. The simple strategy presented here identifies these recombinants by relying on the assumption that recombinants between two adjacent markers are relatively rare events. This strategy has been implemented in the computer program CHROMLOOK, and examples of its use are given. Identification of recombinants allows for the directed regenotyping of suspicious data, the quick mapping of new polymorphisms using recombination minimization, and the development of a meiotic breakpoint map.


Mapping around the Xq13.1 breakpoints of two X/A translocations in hypohidrotic ectodermal dysplasia (EDA) female patients. Cellular hybrids were obtained from a t(X;12) identified in a female patient with hypohidrotic ectodermal dysplasia (EDA). This rearrangement had the same Xq13.1 cytogenetic breakpoint as a t(X;9) found in a previously observed EDA patient. A comparative analysis of these two rearrangements with nine probes was performed at the molecular level. These probes could define three subregions: three are proximal, two are distal, and four are between the two breakpoints. These last probes should prove useful for cloning the gene.


Mapping of bovine markers CYP21, PRL, and BOLA DRBP1 by genetic linkage analysis in reference pedigrees. We have analyzed DNA from 13 bovine reference pedigrees using primers specific for microsatellite markers derived from the 21-steroid hydroxylase (CYP21) and prolactin (PRL) genes and the leukocyte antigen (BOLA DRBP1) pseudogene. Linkage was demonstrated between PRL and BOLA DRBP1 (theta = 0.05; Z = 19.6), cyp21 and PRL (theta = 0.13; Z = 6.8), and BOLA DRBP1 and CYP21 (theta = 0.17; Z = 10.4). These results suggest an order BOLA DRBP1-PRL-CYP21, although in a multilocus analysis the alternative order PRL-BOLA DRBP1-CYP21 was also possible. The data confirm and extend the previously established syntenic relationship between these markers on bovine chromosome 23 and provide points of anchorage for further linkage studies in the reference pedigrees described.


Chromosomal localization of the genes encoding the p50/p105 subunits of NF-kappa B (NFKB2) and the I kappa B/MAD-3 (NFKBI) inhibitor of NF-kappa B to 4q24 and 14q13, respectively. The regulation of expression of a variety of genes involved in immune function, inflammation, and cellular growth control, as well as control of expression of certain viruses such as the human immunodeficiency virus (HIV), is dependent on the transcription factor NF-kappa B. In many cells, NF-kappa B is found in the cytoplasm where it is associated with an inhibitor protein known as I kappa B. Recently the genes encoding the p50 and p65 subunits of NF-kappa B, as well as one form of I kappa B/MAD-3 (NFKBI), have been cloned. As part of our goal to determine the chromosomal organization of members of the REL/NFKB family, as well as their inhibitors, we localized the NFKBp50/p105 (NFKB2) and I kappa B/MAD-3 (NFKBI) genes to human chromosome bands 4q24 and 14q13, respectively.


Physical localization of chromosome 20 markers using somatic cell hybrid cell lines and fluorescence in situ hybridization. A panel of somatic cell hybrid cell lines containing different parts of human chromosome 20 and fluorescence in situ hybridization have been used to physically localize markers to human chromosome 20. Through these complementary approaches and genetic linkage analysis, D20S16, which is closely linked to the maturity onset diabetes of the young (MODY) locus, was mapped to band 20q12 --> q13.1. The gene for growth hormone-releasing factor (GHRF) was physically mapped and reassigned to 20q11, suggesting that GHRF plays no direct role in MODY. In addition, the genes for the chromosome 20-linked glycogen phosphorylase (GYPB) and the bone morphogenetic protein (BMP2A) have been assigned to chromosome 20p, and the interleukin-6-dependent DNA-binding protein (TCF5) has been assigned to 20q12 --> q13 by hybridization to genomic DNA from the panel of somatic cell hybrid cell lines. These approaches are useful for rapid localization of candidate genes for MODY and other DNA markers mapped to chromosome 20.


Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization. "Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.


Mapping of 50 cosmid clones isolated from a flow-sorted human X chromosome library by fluorescence in situ hybridization. Fifty cosmids have been mapped to metaphase chromosomes by fluorescence in situ hybridization under conditions that suppress signals from repetitive DNA sequences. The cosmid clones were isolated from a flow-sorted human X chromosome library. Thirty-eight of the clones were localized to chromosome X and 12 to autosomes such as chromosomes 3, 7, 8, 14, and 17. Although most of the cosmids mapped to the X chromosome appeared to be scattered along both the short and long arms, 10 cosmids were localized to the centromeric region of the chromosome. Southern blot analysis revealed that only two of these clones hybridized to probe pXBR-1, which detects the DXZ1 locus. In addition, 4 out of 5 cosmids mapped on chromosome 8 also localized on the centromeric region. While localization of X-specific cosmids will facilitate the physical mapping of the human X chromosome, cosmids mapped to the centromeric regions of chromosomes X and 8 should be especially useful for studying the structure and organization of these regions.


Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient. The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.


Evidence for the localization of a malignant hyperthermia susceptibility locus (MHS2) to human chromosome 17q. Malignant hyperthermia susceptibility is a lethal autosomal dominant disorder of skeletal muscle metabolism that is triggered by all potent inhalation anesthetic gases. Recent linkage studies suggest a genetic locus for this disorder on 19q13.1. We have previously reported three unrelated families diagnosed with MHS that are unlinked to markers surrounding this locus on 19q13.1. In this report we extend these observations and present linkage studies on 16 MHS families. Four families (25%) were found linked to the region 19q12-q13.2 (Zmax = 2.96 with the ryanodine receptor at theta = 0.0). Five families (31%) were found closely linked to the anonymous marker NME1 (previously designated NM23) on chromosome 17q11.2-q24 (Zmax = 3.26 at theta = 0.0). Two families (13%) were clearly unlinked to either of these chromosomal regions. In five additional families, data were insufficient to determine their linkage status (they were potentially linked to two or more sites). The results of our heterogeneity analyses are consistent with the hypothesis that MHS can be caused in humans by any one of at least three distinct genetic loci. Furthermore, we provide preliminary linkage data suggesting the localization of a gene in human MHS to 17q11.2-q24 (MHS2), with a gene frequency of this putative locus approximately equal to that of the MHS1 locus on 19q.


A radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 locus. We describe a high-resolution radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 (NF2) gene. Eighty-five hamster-human somatic cell hybrids generated by X-irradiation and cell fusion were used to generate the radiation hybrid map. The presence or absence of 18 human chromosome 22-specific markers was determined in each hybrid by using Southern blot hybridization. Sixteen of the 18 markers were distinguishable by X-ray breakage in the radiation hybrids. Analysis of these data using two different mathematical models and two different statistical methods resulted in a single framework map consisting of 8 markers ordered with odds greater than 1000:1. The remaining nonframework markers were all localized to regions consisting of two adjoining intervals on the framework map with odds greater than 1000:1. Based on the RH map, the NF2 region of chromosome 22, defined by the flanking markers D22S1 and D22S28, is estimated to span a physical distance of approximately 6 Mb and is the most likely location for 9 of the 18 markers studied: D22S33, D22S41, D22S42, D22S46, D22S56, LIF, D22S37, D22S44, and D22S15.


Evidence for a third transcript from the human factor VIII gene. Intron 22 of the human factor VIII gene was recently found to contain a gene, associated with a CpG island, which is transcribed in the direction opposite to factor VIII. We now report that another transcript emanates from the island and is transcribed in the same direction as factor VIII. The divergent transcripts originate within 122 bases of each other. The newly identified 5' exon in intron 22 potentially codes for eight amino acids and is spliced to exons 23-26, with the factor VIII reading frame maintained. The protein encoded by this transcript would include the factor VIII C2 domain, responsible for phospholipid binding and essential for coagulant activity.


Systematic detection of errors in genetic linkage data. Construction of dense genetic linkage maps is hampered, in practice, by the occurrence of laboratory typing errors. Even relatively low error rates cause substantial map expansion and interfere with the determination of correct genetic order. Here, we describe a systematic method for overcoming these difficulties, based on incorporating the possibility of error into the usual likelihood model for linkage analysis. Using this approach, it is possible to construct genetic maps allowing for error and to identify the typings most likely to be in error. The method has been implemented for F2 intercrosses between two inbred strains, a situation relevant to the construction of genetic maps in experimental organisms. Tests involving both simulated and real data are presented, showing that the method detects the vast majority of errors.


Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5). A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.


Duplication/deficiency mapping of situs inversus viscerum (iv), a gene that determines left-right asymmetry in the mouse. A recessive mutation in the mouse, situs inversus viscerum (iv), results in randomization of organ position along the left-right body axis: approximately 50% of the progeny of homozygous matings exhibit situs solitus and 50% exhibit situs inversus. Recent studies have established genetic linkage between iv and the immunoglobulin heavy chain gene complex (Igh-C), located on distal mouse chromosome 12. In the present study, we have refined the genetic map location of iv relative to the breakpoint of a reciprocal translocation, T(5;12)31H, involving the telomeric region of chromosome 12 distal to Igh-C and the proximal region of chromosome 5. The translocation results in a large 12(5) derivative chromosome and a small 5(12) derivative chromosome. Because mice with either monosomy or tertiary trisomy for the 5(12) chromosomal region are viable, duplication/deficiency mapping is possible. Deficiency mapping was performed by mating iv/iv homozygotes and T31H heterozygotes. Two animals monosomic for distal mouse chromosome 12 were produced. One of the animals with cytogenetically confirmed monosomy for distal chromosome 12 exhibited situs inversus, indicating that the iv mutation is located at or distal to the T31H breakpoint. For duplication analysis, matings were initially carried out between iv/iv homozygotes and unbalanced T31H animals trisomic for distal chromosome 12. Cytogenetically verified tertiary trisomic progeny were identified and backcrossed with iv/iv homozygotes. The resulting trisomic progeny, 50% of which are expected to carry the iv mutation on both cytogenetically normal copies of chromosome 12, were scored for phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation of DNA markers from a region between incontinentia pigmenti 1 (IP1) X-chromosomal translocation breakpoints by a comparative PCR analysis of a radiation hybrid subclone mapping panel. A strategy based on the use of human-specific interspersed repetitive sequence (IRS)-PCR amplification was used to isolate regional DNA markers in the vicinity of the incontinentia pigmenti 1 (IP1) locus. A radiation hybrid (RH) resulting from a fusion of an irradiated X-only somatic cell hybrid (C12D) and a thymidine kinase deficient (TK-) hamster cell line (a23) was identified as containing multiple X chromosome fragments, including DNA markers spanning IP1 X-chromosomal translocation breakpoints within region Xp11.21. From this RH, a panel of subclones was constructed and analyzed by IRS-PCR amplification to (a) identify subclones containing a reduced number of X chromosome fragments spanning the IP1 breakpoints and (b) construct a mapping panel to assist in identifying regional DNA markers in the vicinity of the IP1 locus. By using this strategy, we have isolated three different IRS-PCR amplification products that map to a region between IP1 X chromosome translocation breakpoints. A total of nine DNA sequences have now been mapped to this region; using these DNA markers for PFGE analyses, we obtained a probe order DXS14-DXS422-MTHFDL1-DXS705. These DNA markers provide a starting point for identifying overlapping genomic sequences spanning the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should assist the molecular analysis of this locus.


A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints. Radiation hybrid mapping was used in combination with physical mapping techniques to order and estimate distances between 14 loci in the proximal region of the short arm of the human X chromosome. A panel of radiation hybrids containing human X-chromosomal fragments was generated from a Chinese hamster-human cell hybrid containing an X chromosome as its only human DNA. Sixty-seven radiation hybrids were screened by Southern hybridization with sets of probes that mapped to the region Xp11.4-Xcen to generate a radiation hybrid map of the area. A physical map of 14 loci was constructed based on the segregation of the loci in the hybrid clones. Using pulsed-field gel electrophoresis (PFGE) analyses and a somatic cell hybrid mapping panel containing naturally occurring X; autosome translocations, the order of the 14 loci was verified and the loci nearest to the X-chromosomal translocation breakpoints associated with the disease incontinentia pigmenti 1 (IP1) were identified. The radiation hybrid panel will be useful as a mapping resource for determining the location, order, and distances between other genes and polymorphic loci in this region as well as for generating additional region-specific DNA markers.


Molecular cloning and characterization of the rat liver IL-6 signal transducing molecule, gp130. Interleukin-6 (IL-6) is a multifunctional cytokine that exerts its effects on different target cells by interacting with a specific receptor. This interaction leads to the association and activation of a second membrane glycoprotein, gp130, which is the IL-6 signal transducing molecule. The nucleotide sequence of gp130 from a human B-cell line has been reported. We report here the cloning and sequence analysis of the gp130 molecule derived from rat liver. Comparison of gp130 molecules from the different species and cell types reveals 78% overall amino acid homology and 94% identity in the growth factor signaling domain. Two gp130 mRNA species, a moderately abundant species of 7.5 kb and a lesser one of 9.0 kb, were present in rat hepatocytes. Ribonuclease protection analyses demonstrated the presence of gp130 mRNA in four different nontransformed cell types: hepatocytes, astrocytes, fibroblasts, and endothelial cells. The sequences between both gp130s in the different cell types are quite similar, supporting the prediction that the different responses initiated by IL-6 on different target cells are modulated by cell-specific proteins distal to the activated gp130 molecule.


A testis-expressed Zn finger gene (ZNF76) in human 6p21.3 centromeric to the MHC is closely linked to the human homolog of the t-complex gene tcp-11. A novel testis-expressed Zn finger gene (ZNF76) was identified by screening cDNA libraries with cosmids derived from 6p21. ZNF76 is a member of the GLI-Krüppel family of DNA binding proteins. It is conserved in mouse where transcription in testis is initiated at Day 20 after birth. The mouse tcp-11 gene is located in the distal inversion of the t-complex and is developmentally regulated in the same manner as ZNF76. The human homolog of tcp-11 was isolated to allow a precise chromosomal localization. By using a combination of somatic cell hybrids, radiation hybrids, metaphase and interphase fluorescent in situ hybridization, and pulsed-field gel electrophoresis, we mapped the two genes to the 6p21.2 to 6p21.3 region and linked them to each other within 300 kb of DNA, approximately 2 Mb centromeric to the major histocompatibility complex.


Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malignant melanoma. Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.


Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35. Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.


Comparison of five tandem repeat loci between humans and chimpanzees. Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.


Linkage studies of Usher syndrome type 1: exclusion results from the Usher syndrome consortium. Usher Syndrome Type 1 is an autosomal recessive disease characterized by profound congenital hearing impairement and vestibular dysfunction followed by the onset of retinitis pigmentosa in childhood or early adolescence. Members of the Usher Syndrome Consortium, whose objective is to locate and isolate the genes for Usher syndrome, have pooled linkage data from 36 families with 111 affected individuals. We report the analysis of 206 blood group, protein, and DNA marker polymorphisms. No evidence of linkage heterogeneity among families was found for any of the markers studied; the negative lod scores exclude the locus for this disease from about 39% of the genome. Our results indicate the regions of the genome to which our continuing efforts should be directed.


A genetic linkage map of human chromosome 9q. A genetic linkage map of human chromosome 9q, spanning a sex-equal distance of 125 cM, has been developed by genotyping 26 loci in the Venezuelan Reference Pedigree. The loci include 12 anonymous microsatellite markers reported by Kwiatkowski et al. (1992), several classical systems previously assigned to chromosome 9q, and polymorphisms for the genes tenacin (HXB), gelsolin (GSN), adenylate kinase 1 (AK1), arginosuccinate synthetase (ASS), ABL oncogene (ABL1), ABO blood group (ABO), and dopamine beta-hydroxylase (DBH). Only a marginally significant sex difference is found along the entire length of the map and results from one interval, between D9S58 and D9S59, that displays an excess of female recombination. A comparison of the genetic map to the existing physical data suggests that there is increased recombination in the 9q34 region with a recombination event occurring every 125-400 kb. This map should be useful in further characterizing the relationship between physical distance and genetic distance, as well as for genetic linkage studies of diseases that map to chromosome 9q, including multiple self-healing squamous epithelioma (MSSE), Gorlin syndrome (NBCCS), xeroderma pigmentosum (XPA), nail-patella syndrome (NPS1), torsion dystonia (DYT1), and tuberous sclerosis (TSC1).


Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification. We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.


A new method for constructing NotI linking and boundary libraries using a restriction trapper. We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.


High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families. Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.


Localization of the gene encoding the alpha subunit of human interleukin-5 receptor (IL5RA) to chromosome region 3p24-3p26. The chromosomal location of the human gene for the alpha subunit of interleukin-5 receptor (IL5RA) has been determined. The human IL5RA gene was localized to the short arm of chromosome 3 by Southern blot analysis of DNA from a panel of mouse-human hybrid somatic cell lines. The IL5RA gene has been further localized to human chromosome region 3p24-3p26 by in situ hybridization of a molecularly cloned IL5RA cDNA fragment to metaphase chromosomes. The results suggest that the IL5RA locus is unlinked to other members of the hematopoietic receptor family. Assignment of the IL5RA gene to chromosome 3 at bands p26-p24 raises the possibility that it may be altered by certain nonrandom chromosomal abnormalities arising in human hematopoietic malignancies and solid tumors.


A bone morphogenetic protein subfamily: chromosomal localization of human genes for BMP5, BMP6, and BMP7. Bone morphogenetic proteins (BMPs) were originally identified by the ability of a demineralized bone extract to induce endochondral osteogenesis in vivo. Seven BMP cDNAs (BMP1 through BMP7) have been recovered through molecular cloning. Recombinant protein products from six of these clones (BMP2 through BMP7) are members of the transforming growth factor beta (TGF-beta) superfamily of regulatory molecules. Based upon a high degree of amino acid sequence homology, BMP5, BMP6, and BMP7 constitute a subfamily within the BMPs. Using human-rodent somatic cell hybrid lines and cDNA probes, we mapped the three members of this subfamily of genes to the human chromosomes. BMP5 and BMP6 are syntenic on human chromosome 6, while BMP7 is syntenic with previously localized BMP2 on human chromosome 20. This analysis reveals that BMP6 maps to a conserved region between the mouse and human genomes. Sequence analysis suggests that the Drosophila 60A gene is the dipteran homolog of this BMP subfamily and may provide clues to the physiologic functions of the products of these genes in human biology.


Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37. A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.


Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization. Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.


Localization of the human KRAB finger gene ZNF117 (HPF9) to chromosome 7q11.2. A cluster of Krüppel type zinc finger genes of the KRAB subclass has recently been localized on human chromosome 19p12-p13.1. We now report that ZNF117 (HPF9), a closely related zinc finger gene of this KRAB subfamily, has been assigned to a distinct locus in the human genome: chromosome band 7q11.2.


The gene for tissue inhibitor of metalloproteinases-2 is localized on human chromosome arm 17q25. Tissue inhibitor of metalloproteinases-2 (TIMP2) is a natural inhibitor of several proteinases that are involved in the degradation of the extracellular matrix. By means of somatic cell hybrids segregating human chromosomes, the gene encoding this inhibitor was assigned to human chromosome 17. Fluorescence in situ hybridization confirmed this assignment and allowed mapping of the gene to the terminal region (17q25) of the chromosome.


Autosomal localization of the amelogenin gene in monotremes and marsupials: implications for mammalian sex chromosome evolution. We have determined by Southern blot analysis that DNA sequences homologous to the AMG gene probe are present in the genomes of both marsupial and monotreme mammals, although adult monotremes lack teeth. In situ hybridization and Southern analysis of cell hybrids demonstrate that AMG homologues are located on autosomes. In the Tammar Wallaby, AMG homologues are located on chromosomes 5q and 1q and in the Platypus, on chromosomes 1 and 2. The autosomal location of the AMG homologues provides additional support for the hypothesis that an autosomal region equivalent to the human Xp was translocated to the X chromosome in the Eutheria after the divergence of the marsupials 150 million years ago. The region containing the AMG gene is therefore likely to have been added 80-150 million years ago to a pseudoautosomal region shared by the ancestral eutherian X and Y chromosome; the X and Y alleles must have begun diverging after this date.


Assignment of a novel protein tyrosine phosphatase gene (Hcph) to mouse chromosome 6. Hematopoietic cell phosphatase (Hcph) was identified by amplification of conserved protein tyrosine phosphatase sequences from a myeloid cell line and is predominantly expressed in hematopoietic cells. Hcph is unique in containing two, tandemly repeated, src-homology 2 domains in the amino terminal region of the phosphatase. Using a genomic probe in interspecific backcross analysis, the murine Hcph gene maps to mouse Chromosome 6 and is tightly linked to the Tnfr-2 and Ly-4 genes.


Identification of a processed pseudogene related to the functional gene encoding the GM2 activator protein: localization of the pseudogene to human chromosome 3 and the functional gene to human chromosome 5. The GM2 activator protein is an essential substrate cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A (EC 3.2.1.52). There have been conflicting reports as to the chromosomal localization of the gene encoding the activator. We demonstrate here that these conflicts were caused by the presence of a previously unidentified processed activator-pseudogene on chromosome 3, and we confirm a previous ELISA-based localization of the functional activator gene to chromosome 5. Our data indicate that the functional activator locus can still be considered a candidate site for defects causing some forms of spinal muscular atrophy.


Autosomal dominant retinitis pigmentosa: a novel mutation at the peripherin/RDS locus in the original 6p-linked pedigree. Using single-strand conformation polymorphism electrophoresis, heteroduplex analysis, and direct sequencing, we have searched for possible disease-causing mutations in the adRP family in which we originally found tight linkage of the disease to 6p. We have now identified a single base change in exon 2, which results in the replacement of a serine residue at codon 212 for a glycine residue. The mutation cosegregates with the disease with a lod score of 12.1 at theta = 0.0.


Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels. We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.


Evidence for nonallelic genetic heterogeneity in autosomal recessive retinitis pigmentosa. Recent evidence suggesting the involvement of mutant rhodopsin proteins in the pathogenesis of autosomal recessive retinitis pigmentosa has prompted us to investigate whether this form of the disease shows non-allelic genetic heterogeneity, as has previously been shown to be the case in autosomal dominant retinitis pigmentosa. The availability of a unique inbred Dutch pedigree has enabled us to address this question. We have used an intragenic polymorphism to exclude the possibility that a mutation in the rhodopsin gene is responsible for the disease in this patient population. These data provide evidence for the involvement of at least two loci in autosomal recessively inherited retinitis pigmentosa.


Dinucleotide repeat (GT)n markers on chromosome 21. To further develop the linkage map of human chromosome 21 (HC21), we have concentrated on identifying highly polymorphic markers based on dinucleotide repeat sequences such as (GT)n, as these are often highly polymorphic, are widespread throughout the human genome, and can be rapidly analyzed by the polymerase chain reaction. We report here nine (GT)n polymorphic markers from HC21.


VH-gene representation in autoantibodies reflects the normal human B-cell repertoire. The recurrence in the V-gene repertoire of individual germline VH genes can now be extended from the restricted B-cell populations of the fetus, autoantibodies and B-cell malignancies to the expressed V-gene repertoire of normal adults. Why the human B cell preferentially utilizes these individual VH genes remains speculative. However, it is apparent that the population of VH genes used to encode autoantibodies reflects the normal expressed repertoire (Fig. 7). Even so, the overrepresentation of other V genes such as Dxp'1 in anti-DNA antibodies and the presence of somatic mutation in the pathogenic autoantibodies of autoimmune disease continues to suggest an antigenic influence on V-gene selection. We postulate that only a fraction of available germline V genes are utilized in the expressed repertoire, and that polyspecificity of naturally occurring antibodies and somatic mutation of CDR3 compensate for the loss of diversity entailed by the limited use of the potential repertoire. The mechanisms by which germline genes become pathogenic remains unclear but they presumably relate to mutation, loss of regulatory control or perhaps environmental factors (Isenberg et al. 1992). What then are the mechanisms which lead to escape of these VH genes from normal control? What antigenic drive if any produces anti-DNA specificity in SLE? Why indeed is the expressed repertoire using only a fraction of the available germline? To answer these questions, further study of the V-gene repertoire of selected populations of antigen-binding cells and of pathogenic IgG autoantibodies is necessary and is ongoing. The contribution of individual V genes to antigen binding and idiotype is also being dissected and promises to yield important information about the relative contribution of VH genes to autoimmunity.


Selection of immunoglobulin variable regions in autoimmunity to DNA. Results from our analyses of variable region gene usage among spontaneous anti-DNA antibodies in autoimmune mice have indicated that both the early IgM and later-appearing IgG autoantibodies to DNA are generated by clonally selected B cells. The recurrent usage of particular variable region genes among all the anti-DNA hybridomas analyzed and reported to date supports this hypothesis. The preferential expression of particular light and heavy chain variable region genes among selected populations of both IgM and IgG anti-DNA hybridomas likewise supports the hypothesis. Both IgM and IgG antibody-producing B cells are derived from the same clonal precursor population and may be derived from the same B cell clonal precursor within an individual mouse. The selective and recurrent expression of germline and somatically-derived structures that would be expected to promote protein binding to DNA within anti-DNA antibody variable regions, particularly arginines in both light and heavy chain complementarity-determining regions, indicates that DNA or DNA-containing complexes may be the antigen that stimulates anti-DNA antibody in autoimmune mice. The progressive increase in the specificity of spontaneous anti-DNA antibodies for native DNA as the autoimmune response matures from IgM to IgG likewise suggests that DNA may be the antigenic stimulus for spontaneous anti-DNA in autoimmune mice. A hypothetical, computer-generated model of anti-DNA antibody binding to DNA provides an interesting paradigm for the molecular basis of antibody specificity for DNA.


Antegrade and retrograde cardioplegia with different reperfusion techniques in patients with multiple coronary artery lesions. To evaluate the effectiveness of retrograde cardioplegia and reperfusion, a total of 266 patients undergoing coronary bypass surgery between Nov 1987 to Dec 1989 were divided into three groups depending on the method of cardioplegic fluid delivery and reperfusion. In group I (80 patients) antegrade cardioplegia and reperfusion was used. In group II (98 patients) antegrade and retrograde cardioplegia and antegrade reperfusion was used while in group III antegrade and retrograde cardioplegia and retrograde reperfusion was used. Myocardial functions were studied with the help of an on-line computer on the basis of mathematical model of heart before and after cardiopulmonary bypass. Biopsy specimens were collected before, during and after cardiopulmonary bypass in order to study myocardial structural changes. In group I patients there was decrease in myocardial function in the immediate post perfusion period while group II patients had considerable improvement in their myocardial function and groups III patients showed further improvement in it. Ultrastructural myocardial study revealed considerable detrimental changes in group I, minimal changes in group II and no change in group III patients. Thus in our experience retrograde cardioplegia and retrograde reperfusion with warm oxygenated blood provide maximum myocardial protection in patients with multiple coronary artery lesions.


Cannulation of the internal jugular vein in patients undergoing open heart surgery. A total of 476 internal jugular vein cannulations performed between June 1990 to August 1991 were prospectively evaluated. The patients' age ranged between ten days to 61 years. We achieved a high success rate of cannulation (97.9%). We describe the use of two single lumen catheters inserted through right internal jugular vein as an alternative to the popular multilumen catheters. We also suggest that low approach should be tried more often in children, if the central approach fails.


High risk coronary angioplasty using percutaneous cardiopulmonary bypass support. Twenty high risk patients with severe angina were subjected to balloon angioplasty after instituting percutaneous cardiopulmonary bypass support to enhance the safety of high risk elective coronary angioplasty. All patients had a low ejection fraction, a large amount of viable myocardium perfused by the targeted artery or both (left ventricular ejection fraction < or = 25% in 15 patients). Three vessel disease was present in all. Angioplasty of the only remaining vessel was done in 14 patients, 2 vessels in 5 patients and a sequential graft in 1 patient. Bypass flows ranged from 2.8-4.5 litres. Bypass was discontinued after a mean bypass time of 35 min. Haemostasis was achieved by external clamp compression in 16 patients. The angioplasty was successfully performed in all the patients and the procedure was well tolerated. During the bypass period the pulmonary artery diastolic pressures ranged from 0-8 mm Hg. There was 1 hospital death due to abrupt vessel closure. Two patients required surgical help to repair femoral artery. During the follow up period of 1-12 months, 67% patients have no angina and only 1 has died. Our experience demonstrates the safety and efficacy of percutaneous bypass support in selected patients undergoing high risk coronary angioplasty.


Percutaneous transluminal coronary angioplasty in patients over the age of 65 years. PTCA performed in 672 patients (646 patients < 65 years and 26 patients > or = 65 years) from March 1986 to September 1990 consisting of 619 males and 53 females are being discussed for the purpose of comparison of the results between patients in the < 65 years and > or = 65 years age groups. It was found that diabetes mellitus (50 vs 24.4.%, P < 0.01), obesity (46.1 vs 26%, P < 0.05), and hyperlipidaemia (66 vs 37.1%, p < 0.01) had a higher incidence among the elderly. Of the total 1047 lesions observed, 662 lesions in the < 65 years age group and 37 lesions in the > or = 65 years age group were dilated by PTCA (1.02 lesions/patient and 1.4 lesions/patient respectively). The lesion morphology showed higher degree of irregular contour in the < 65 years age group (23.6 vs 8.1%, p < 0.05), though the number of lesions with PTCA attempted in all age groups were almost similar. Major complications included one MI (3.8%) and two deaths (7.6%) in the > or = 65 years age group. During followup (mean duration: 8.2 months > or = 65 years and 13.5 months < 65 years age group) significantly higher percentage of patients < 65 years were asymptomatic when compared to elderly (50.2 vs 15.4% p < 0.05). Angina status was same more often in the elderly (23.1 vs 2.8% p < 0.01). The cumulative rate for repeat PTCA was similar for elderly and younger patients.


Long term results of percutaneous transluminal valvuloplasty in patients with valvular aortic stenosis. The results of percutaneous balloon aortic valvuloplasty (PBAV) in 62 consecutive patients with valvular aortic stenosis are reported. The age of the patients ranged from 11 months to 72 years (mean 28 +/- 12 years). Hemodynamically successful dilatation was achieved in 58 out of 62 patients. This was associated with marked clinical improvement in these patients. The left ventricular aortic peak to peak gradient decreased from 96.67 +/- 38.4 to 28.14 +/- 26.5mmHg (p < 0.01). There were no deaths during the procedure. Only one patient died in the hospital during the same admission. There was an increase in aortic regurgitation (AR) by at least one grade in 25 (40.3%) patients. Femoral arterial thrombosis was seen in 9/62 patients, 5 of them requiring surgical intervention. Follow up was available in 28 (45.1%) patients over a period of 2-15 months (mean 9 +/- 3 months). Two patients died during the follow up period. Doppler evaluation of gradients was done in all 28 patients with 15 consenting to undergo repeat cardiac catheterisation. Although hemodynamically the restenosis rate was 35.7% (10/28), only 2 of these patients showed symptomatic deterioration. The success of dilatation and restenosis rate were independent of the etiology of aortic stenosis, presence of calcification and the number of balloons used. This study demonstrates that PBAV is feasible in valvular aortic stenosis at low risk and is able to produce significant clinical and hemodynamic improvement in most cases with a restenosis rate of 35.7% at a follow up period of 9 +/- 3 months.


Acute hemodynamic effects of different atrioventricular intervals in dual chamber pacemakers: is there an optimum atrioventricular delay. Conflicting data have been reported regarding adjustment of atrioventricular (AV) interval to maximise hemodynamic performance of dual chamber pacemakers. Eleven consecutive patients with complete heart block and dual chamber pacemakers were paced at three AV intervals (150, 200, 250 msec) and free running rates (60-93 bpm, mean 73 +/- 12 bpm) with simultaneous measurements of cardiac output, atrial contribution to left ventricular filling, left ventricular ejection fraction, and peak aortic velocity and acceleration by echo-Doppler techniques to define the optimum AV delay. At all the three AV intervals tested there was no difference in cardiac output (4.7 +/- 0.96, 4.83 +/- 1.12, 4.77 +/- 1.19 litres/min respectively, p = NS), left ventricular ejection fraction (60.2 +/- 10.6%, 61.2 +/- 9.9% and 64 +/- 8.3%, p = NS), atrial contribution to left ventricular filling (0.37 +/- 0.10, 0.38 +/- 0.09, 0.36 +/- 0.16, n = 8, p = NS), peak aortic velocity (104 +/- 8, 105 +/- 12, 104 +/- 13 cm/sec, p = NS) and aortic acceleration (19.68 +/- 4.26, 20.4 +/- 5.58 and 19.0 +/- 4.54 m/sec2, p = NS). Compared to AV delay of 150 msec an increase in cardiac output of 0.5 L/minute was observed in three patients at an AV delay of 200 msec and in one patient at the AV delay of 250 msec. These data suggest that it is difficult to generalize an optimum AV delay in patients with dual chamber pacemakers. With the AV interval in the range of 150-250 msec, only a minority of patients could improve their haemodynamics at rest by adjusting this interval if the baseline cardiac function was normal.


Experience with recycled cardiac pacemakers. Twelve years experience with pacemaker re-use is reported. Between November 1979 and December 1991, forty two pacemakers were re-used in 42 patients after in-hospital reconditioning. There were 31 males and 11 females in the age range of 29 to 84 years (mean 59 +/- 13 years). In 29 patients, the pulse generator was explanted because of pocket infection and/or impending extrusion, and it was re-implanted at a new site after sterilization. In the remaining 13 patients pacemakers explanted from other patients were implanted because of economic reasons. All patients were regularly followed up in the pacemaker clinic for a mean duration of 34 +/- 35 months (range 6-126 months). There was one infection and one erosion after 12 and 27 months respectively. One of these units was resterilized and implanted without any recurrence of infection or erosion over a follow up of 12 months. There was no adverse reaction or pulse generator failure. Our experience suggests that the use of refurbished pacemakers is a safe and economically viable option.


Modified cephalic vein guide wire technique for permanent pacemaker implantation. This report describes a modified cephalic vein guide wire technique used for implantation of 18 consecutive pacemakers using bipolar leads. The modified technique appears to be a safe and simple procedure for single chamber permanent pacing. Its use may also be extended in implanting dual chamber pacemakers.


Coronary artery disease in premenopausal Indian women: risk factors and angiographic profile. Coronary angiographic and clinical profile of 47 premenopausal women presenting with myocardial infarction (MI) or angina is presented. Seventeen patients (36%) had significant obstructive coronary artery disease (CAD) (Group I), while 30 (64%) had normal coronaries (Group II). The latter group included 4 who had MI and 26 who presented with angina. Risk factors in Group I included hypertension (53%), diabetes mellitus (24%), hypercholesterolemia (29%), oral contraceptives and a positive family history (11.8%). Frequency of one, two and three vessel disease was 47%, 18% and 35% respectively. The left anterior descending artery was most commonly affected (82%). In Group II the risk factors included hypertension (17%) and diabetes (7%). No patient in either group was a smoker. This analysis shows that significant obstructive CAD in premenopausal Indian females is more commonly associated with hypertension, diabetes and hypercholesterolemia. Smoking was not encountered and ingestion of contraceptive pills is uncommon.


Asymptomatic coronary heart disease detected on epidemiological survey of urban population of Delhi. A community based epidemiological study of coronary heart disease (CHD) was carried out in a random sample of 13723 adults in the age group of 25-64 years in the urban population of Delhi. The electrocardiogram (ECG) of all clinically detected CHD cases and of a sample of 5621 persons (selected on the basis of alternate household screened) without clinical manifestations of CHD, was obtained. Out of 5621 persons labelled as asymptomatic, CHD evidence of Q wave myocardial infarction (MI) was present in 80 ECGs (1.4%). Another 296 ECGs had ST & T changes vide Minnesota Code 4-1-1, 4-1-2, 5-1 and 5-2 acceptable as evidence of probable CHD. The overall prevalence rate of asymptomatic CHD was 6.7% (male 5.6%, female 7.6%). Silent MI was more common in the male patients (1.7% vs 1.1%, p < 0.001). However, ST-T changes were more common in female patients (6.5% vs 3.9%, p < 0.001). The ST-T changes showed a steady factor in asymptomatic CHD cases was hypertension in both sexes (male-45.2%, female-43.5%) p = NS. Obesity was present in 24% of male & 46.1% of female patients (p < 0.001). Family history was found in 20% cases of both sexes. Smoking was recorded in 34.9% male and 10.9% female patients with asymptomatic CHD (p < 0.001).


Feasibility of thrombolytic therapy--a one year prospective study. A one year prospective study was conducted on all the patients admitted with clinical diagnosis of acute myocardial infarction (AMI) to determine the proportion of patients who can be given thrombolytic therapy. Factors responsible for non-administration were analysed. 213 patients with AMI entered the study. Standard inclusion criteria for thrombolytic therapy were used. 101 (47%) patients failed to meet the inclusion criteria. This included 7 (3.3%) who failed to satisfy the electrocardiographic (ECG) criteria. Nine patients with atypical symptoms were unable to reach within the stipulated 6 hours while the remaining 85 (40%) patients were delayed inspite of typical features due to inability of the patient to attribute the symptoms to the heart, lack of proper transport facility and self medication. 112 patients (53%) met the inclusion criteria but 7 patients were excluded because of age (> 70 years), and another 8 because of contraindications. Of the remaining 97 patients, 47 failed to receive thrombolytic therapy due to lack of awareness of the benefits of thrombolytic therapy by the first treating physician, misinterpretation of ECG, inability to afford and refusal to give consent. Only 50 patients (23%) received thrombolytic therapy. This low figure can be easily improved upon by the correction of a number of remediable factors.


Early experience with coronary stenting. From April 1988 to December 1991, we implanted 75 coronary stents (29 self-expanding and 46 balloon expandable) in 62 patients. All had New York Heart Association class II to IV angina, and 11 (18%) had prior coronary artery bypass grafting (CABG). Thirty nine patients (63%) had 1 vessel disease, and 23 (37%) had multivessel disease. The mean left ventricular ejection fraction was 63 +/- 11%. The indications for coronary stenting were acute post percutaneous transluminal coronary angioplasty (PTCA) occlusion in 45 (73%) (bail out stenting) and restenosis in 17 (24%) (elective stenting). There were 52 single stent (84%), 7 double stent (11%), and 3 triple stent procedures (5%). The mean stent diameter was 3.8 +/- 0.5 mm, and the mean stent length 21 +/- 7 mm. The attempted vessels were the left main coronary artery in 2(3%), left anterior descending coronary artery in 27 (44%), left circumflex coronary artery in 8 (13%), right coronary artery in 17 (27%), and a saphenous vein graft in 8 (13%). Technical success was achieved in 74 stent implantations (98%). Technical failure occurred in 1 case with a self expanding stent because of inability to reach the lesion. In hospital complications (mean hospital stay 10 +/- 10, range 2-60 days) included temporary stent occlusion in 2 patients (3%) treated by balloon dilatation and thrombolysis with intravenous urokinase, permanent stent occlusion in 5 patients (8%), Q-wave infarction in 5 patients (8%), CABG in 4 patients (11%), and death in 3 patients (5%). At least 1 major complication (Q wave infarction, CABG, or death) occurred in 8 patients (13%).(ABSTRACT TRUNCATED AT 250 WORDS)


Colour Doppler echocardiographic quantification of prosthetic aortic valve regurgitation in patients with normally functioning Bjork-Shiley prostheses. Colour Doppler echocardiographic studies were performed in 46 patients (age range 16-35 years, mean 26 +/- 8; male 31, female 15) with normally functioning Bjork-Shiley prostheses in aortic position to estimate transprosthetic regurgitation. Regurgitant jet length and height were measured and assessed in multiple views. All patients showed prosthetic regurgitation of varying degree. Regurgitant jets were central in all but 3 (6.5%) patients. Single jets were seen in 28 (61%) and double jets in 18 (39%). Jet height in parasternal long axis view ranged from 0.4 to 1.2 cm (mean 0.7 +/- 0.4 cm) and jet height to left ventricular outflow tract diameter ratio was 0.22 to 0.48 (mean 0.38 +/- 0.13). Prosthetic regurgitation was < or = 2/4 grades in 42 (91%) patients, and combined height of double jets (n = 18) was less than that of the single jets (n = 28) (0.5 +/- 0.3 cm vs 0.8 +/- 0.4 cm, p < 0.05). In conclusion, colour Doppler examination frequently detects prosthetic regurgitation in patients with aortic Bjork-Shiley prostheses; regurgitation is grade 2/4 or less in most of the patients, is overestimated in patients with a single jet and weakly correlates with prosthesis size.


Neonatal pulmonary flow pattern--a pulsed Doppler echo study. The pulmonary flow pattern was analysed by pulsed Doppler in 77 normal neonates, at < 48 hours (36 subjects), 49 to 96 hours (25 subjects) and 97 to 148 hours (16 subjects). From the flow velocity spectrum, using an built in computer system, the acceleration time and the ejection time were measured, and the ratio was calculated. This ratio was 0.24 +/- 0.08 in the subjects within 48 hours whereas this ratio was 0.53 +/- 0.11 in the subjects above 97 hours; thus the ratio increased with age. We noticed that the ratio obtained from the pulsed Doppler is useful in assessing the pulmonary vascular status, even in neonates.


Two dimensional and Doppler echocardiographic evaluation of double chambered right ventricle. Two-dimensional and Doppler echocardiographic findings in 20 patients with double chambered right ventricle are described. All patients had the diagnosis established by cardiac catheterisation and confirmed at operation. Echocardiographic evaluation was done prior to surgical correction. Anomalous muscle bands in right ventricular cavity were detected in 16 patients. Doppler flow velocities in the right ventricular cavity suggested infundibular obstruction to blood flow at a low level in all 17 patients studied by Doppler echocardiography. Ventricular septal defects (11 patients), pulmonary stenosis (3 patients), and aortic regurgitation (3 patients) were detected accurately before operation by echocardiographic examination. Failure to detect the anomalous muscles in right ventricular cavity may occur in adult patients with poor anterior resolution and in those with severe right ventricular outflow obstruction and myocardial hypertrophy. Two dimensional echocardiography with Doppler flow analysis is useful in the evaluation and differential diagnosis of right ventricular outflow obstructions prior to invasive studies and surgical intervention.


Long term performance of silicone insulated and polyurethane insulated cardiac pacing leads. The long term performance of various pacing leads in use for at least one year is reported from one centre. Between January 1975 and December 1990, 1056 cardiac pacing leads were implanted in 881 patients (mean age 57 +/- 13 years). Eight hundred and three leads were silicone insulated (SI) and 253 leads were polyurethane insulated (PUI). Leads from different manufacturers were used (Medtronic, CPI, Telectronics). The average duration of follow up was 56.6 +/- 77 months (range 12-221 months) for SI and 47.1 +/- 24 months (range 12-99 months) for PUI leads. Overall lead failure occurred more often in the PUI group (n = 46, 18.2%) compared to SI group (n = 93, 11.3%, p < 0.025). Majority of lead failures occurred in the first 36 months after implantation. In particular, Medtronic models 6971, 6972 (urethane) and 6901 (silicone) showed higher rate of complications (25.5%, 26.3% and 44.5% respectively).


The efflux of nor-epinephrine from platelets in genetic hypertension. Platelets have been used as a model of sympathetic neurons to study the storage of nor-adrenaline in normotensive individuals belonging to families with essential hypertension for at least two generations. The efflux and initial efflux rate (K) of noradrenaline was determined in 36 young relatives (mean age 29.2 years) and in 20 young controls with no family history of hypertension (mean age 26.5 years). From the groups of relatives all those with definite hypertension had been excluded. Efflux and efflux rate K was significantly higher in young relatives (50.82 +/- 3.84 and 28.2 +/- 6.3) than in the controls (39.2 +/- 0.71 and 15.2 +/- 4.8). Of the relatives 25.2% had higher K values than any of the controls.


Use of captopril as an isolated agent for the management of stable angina pectoris--a double blind randomised trial. In this double blind randomised placebo controlled study, we investigated the antianginal efficacy of oral captopril in 33 patients of angiographically documented coronary artery disease (chronic stable angina). Apart from sublingual nitrates, all other antianginal drugs were withdrawn. Patients were then evaluated both subjectively by questionnaire and objectively by treadmill stress test. No patient had more than mild hypertension and all patients had good left ventricular function. One group of patients received oral captopril while the other group was given placebo. A repeat assessment was done after six weeks and the results compared with baseline. Anginal attacks decreased from 20.11 +/- 1.86 per week on placebo to 9.92 +/- 1.38 (p < 0.01) on captopril as also the number of sublingual nitrates (18.84 +/- 3.01 to 11.14 +/- 2.94, p < 0.01). Assessment by the treadmill stress test showed that in comparison to the pretreatment test, captopril therapy resulted in a significantly increased exercise duration (6.26 +/- 0.21 to 6.98 +/- 0.31 minutes, p < 0.05), total work done (6.76 +/- 0.26 METS to 7.48 +/- 0.29 METS, p < 0.05). In addition there was a significant increase in time to angina (6.16 +/- 0.18 to 6.85 +/- 0.24 min, p < 0.05) and time to 1mm ST depression (5.18 +/- 0.26 to 6.46 +/- 0.30 min, p < 0.01). We conclude that captopril is an effective monotherapy for patients with chronic stable angina and has both antianginal as well as anti-ischemic effects, possibly secondary to direct coronary vasodilation.


Molecular mechanics simulation of the interaction of estrogen receptor with estrogen regulatory element. A model for the interaction of 31 amino acid fragment (protein) from DNA binding domain of human estrogen receptor (hER) with a five base pair DNA sequence 5'GGTCA 3' from estrogen regulatory element (ERE) has been obtained using a step-wise procedure based on structural data on model peptides, DNA binding domain of hER, steric constrains imposed by tetrahedral coordination of the Cys sulphurs with zinc ion and classical secondary structural elements. Structure of the protein as well as its complex with DNA is obtained by energy minimization followed by refinement by molecular mechanics. The complex is stabilized by H-bonds between Lys22, Lys26 and Arg27 with DNA bases G2, T3 and T6. Lys22 also made H-bond with the backbone of G2. The backbone of Cys18 H-bonded with N7 of G1. DNA was in distorted B form and showed evidence of protein-induced conformational changes.


Mechanical ventilation after open heart surgery. One hundred adult patients, undergoing elective open heart surgery over a period of 4 months, were studied to assess the practice of ventilation in the post operative period. The anaesthetic technique employed used moderate doses of morphine, supplemented with halothane and a muscle relaxant. The decision to extubate was based on clinical assessment, and satisfactory blood gases following a 45 minute T-piece trial. The patients were ventilated for an average duration of 8 hours and 2 minutes and 59 out of 100 patients were extubated within 8 hours. Patients undergoing coronary artery bypass graft were ventilated for significantly longer durations (10 hours 28 minutes) (p < 0.05) and had significantly lower arterial oxygen tension (p < 0.01) 30 minutes after extubation, as compared with those undergoing valvular surgery. Also patients whose bypass time exceeded 2 hours had significantly longer extubation times (p < 0.05) as compared with those who had a bypass time less than 1 hour. T piece trial was found to be a satisfactory method of weaning in all the patients.


Computer aided study of the interaction of thyroid hormone receptor with DNA. Interaction of the first zinc finger from human thyroid hormone receptor (finger) with hexanucleotide duplex d(AGGTCA).d(TGACCT) from thyroid regulatory element has been studied by molecular mechanics simulation technique. The structure of the finger as well as its complex with DNA is optimized using constrain of tetrahedral coordination of the zinc ion to Cys sulphurs. The finger is stabilized by series of H--bonds (5 in backbone, 2 in side chains and 2 between backbone and side chains). The complex is stabilized by H-bonds between side chains of Tyr 11, His 12, Tyr 13 and Arg 14 with G2, G3 and G8. DNA is in B form. H--bonding network within DNA is preserved. Opposite strand P-P and Cl'-Cl' distances are changed slightly. There is a systematic change in the backbone torsional angles and sugar pucker. Also, there is an evidence of protein-induced conformational change in DNA.


The role of cadmium in induction of atherosclerosis in rabbits. Experiments were carried out in rabbits to determine the effects of prolonged treatment of cadmium (8 mg/kg/day) for a period of 6 months on histopathological changes and biochemical alterations of lipid profiles in various tissues compared to normal rabbits. No ECG changes were observed before and at the end of cadmium treatment. Histopathological studies of the coronary artery revealed atherosclerotic changes. Total lipids, cholesterol, free fatty acids and phospholipids were significantly increased in heart and kidney, but decreased in serum and liver. Triglyceride content was increased significantly in heart and kidney with a significant depletion in liver and serum. It is postulated that atherosclerotic changes in rabbits probably occurred through toxic effects of cadmium but the exact mechanism needs to be elucidated.


Liposomal hamycin in the control of experimental aspergillosis in mice: relative toxicity, therapeutic efficacy and tissue distribution of free and liposomal hamycin. Therapeutic efficacy of liposomal Hamycin has been evaluated in an animal model system for aspergillosis in Balb/c mice. Hamycin was intercalated into soya phosphatidyl choline (SPC), SPC: choline (1:1, vol./vol.) and DMPC liposomes. A single dose of either 0.1 mg/kg, 0.25 mg/kg or 0.5 mg/kg of liposomal Hamycin and 0.1 mg/kg of free Hamycin was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in the survival rate of animals along with decrease in fungal count in various organs was observed with liposomal administration. Incorporation of cholesterol into liposomes decreased the in vivo toxicity of Hamycin in a dose dependent manner. However, antifungal activity both in the presence and absence of cholesterol showed marked variation as compared to that of non-aromatic polyenes, e.g. amphotericin B. Analysis of Hamycin distribution by HPLC in various tissues revealed higher blood concentration of this drug, when given in free form, compared to its liposomised form. These studies suggest that liposomal Hamycin is more effective than free Hamycin in controlling the experimental Aspergillosis.


Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme. Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.


Lipid composition of thylakoid membranes of cadmium treated wheat seedlings. Cadmium (200 ppm) applied through the rooting medium to 30-day-old wheat plants decreased chlorophyll content, net CO2 exchanges and PSII activity by 34, 54 and 43% respectively. Thylakoid total lipids, total glycolipids, total phospholipids and total neutral lipids decreased by 22, 23, 12 and 25%, respectively, under cadmium treatment. Thylakoid membrane glycolipids had three major constituents, viz. monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol contents decreased by 32 and 27%, respectively, under cadmium. Cadmium application also decreased the concentration of phosphatidyl glycerol and phosphatidyl choline to the extent of about 57 and 31%, respectively. On the other hand, phosphatidic acid and free fatty acids content showed an increase. These compositional changes in thylakoid membranes might be responsible for reduced PSII activity and rate of photosynthesis as observed under cadmium treatment.


Studies on phospholipids of different mutants of Salmonella minnesota. Lipid composition of the cells of smooth (S form) and core-defective rough mutants (Ra, Rb & Re) of Salmonella minnesota has been studied. The readily-extractable lipids (RELs), acid-extractable lipids, and polar and nonpolar phospholipids have been analysed. Fatty acid composition of the different fractions containing phospholipids and other neutral lipids have been determined by GLC and GC-MS techniques. Phosphatidyl glycerol (PG), phosphatidylethanolamine (PE) and diphosphatidyl glycerol (DPG) were the major phospholipids present in all the strains. The major saturated fatty acid found was C16:0, and unsaturated fatty acids were, C16:1 and C18:1. Cyclopropane fatty acids, C17cy and C19cy, were also present in small amounts. Increased amounts of REL and unsaturated fatty acids were found in the mutants compared with the smooth strain. The amount of PG and PE decreased and DPG increased in the mutant strains.


Effect of quassin on the metabolism of catecholamines in different life cycle stages of Culex quinquefasciatus. Quassin, a mosquito larvicide isolated from Quassia amara, inhibits tyrosinase activity in the larvae of Culex quinquefasciatus. Since tyrosinase is directly involved in sclerotisation of the cuticle, it is suggested that quassin, as a larvicide, inhibits development of the cuticle. In presence of quassin phenylalanine, tyrosine and L-dopa levels were increased in larvae. In the larval stages, mosquitoes have a high concentration of phenylalanine and tyrosine with the level of the latter being very high just before pupation and then declines sharply. Monoamine oxidase (MAO), an enzyme directly involved in the metabolism of catecholamines, remained unaffected by quassin, in fact the level of adrenaline also remained unchanged in larvae during quassin poisoning. MAO showed high variation in its activity between synthetic and natural substrates. Tyramine is not a substrate for MAO. Tyrosinase activity was high in developing stages and negligibly low in adults and showed specificity to L-dopa. Phenylalanine and tyramine are unaffected by tyrosinase. Blood feeding did not influence the activity of both these enzymes.


Restoration of antioxidants in liver by methionine feeding in experimental rat urolithiasis. The effect of methionine or citrate on antioxidant defense system has been studied in urolithic rat. Liver weight and its protein concentration did not change in the rats fed with calculi producing diet (CPD) when compared to normal diet fed rats. Feeding rats along with citrate (c-CPD) or methionine (m-CPD) improved their body weight gain. Liver microsomes and mitochondria fractions of CPD and c-CPD fed groups showed increased susceptibility for lipid peroxidation in presence of ascorbate and t-butyl hydroperoxide when compared to either control or m-CPD fed groups. Increased superoxide dismutase and xanthine oxidase activities, decreased catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, decreased concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin-E and increased formation of hydroxyl radical, hydroperoxides and diene conjugates were observed in the liver of both CPD fed group as well as c-CPD fed group. Except SOD and xanthine oxidase, all other parameters were normalized in m-CPD fed group. This suggested that feeding methionine reduced the susceptibility for lipid peroxidation by restoration of the level of free radical scavengers.


Role of antibiotics in lipid peroxidation. Alterations in the levels of lipid peroxides, superoxide dismutase, catalase, glutathione, free fatty acid and serum ceruloplasmin were studied in rats fed with high fat cholesterol diet administered different antibiotics, viz. ampicillin, tetracycline, streptomycin, chloramphenicol and cephalosporin. The concentrations of lipid peroxides, glutathione, free fatty acid decreased in most of the tissues, except in tetracycline, streptomycin and cephalosporin treated rats. The changes observed in the activities of superoxide dismutase and catalase in the liver and kidney of these antibiotics administered groups also are in accordance with the changes in lipid peroxides. The results show that the tetracycline is hepatotoxic and nephrotoxic, while cephalosporin and streptomycin are nephrotoxic.


Effect of aspirin on isoproterenol induced myocardial infarction--a pilot study. Effect of aspirin (1.2 mg/100 g body wt orally for 30 days) on myocardial infarction induced by isoproterenol (200 mg/kg body wt, subcutaneously for 2 days) has been studied in rats using activities of creatine kinase, aspartate amino transaminase, alanine amino transaminase and lactate dehydrogenase and levels of lipid peroxides as standard markers. Aspirin treatment is found to counteract the effect of isoproterenol on lipid peroxide formation and associated enzyme changes in serum and heart.