Somatic Cell Hybrid Mapping Research Articles

Human-rodent somatic cell hybrid mapping panels

Human-rodent somatic cell hybrid mapping panels

Overview of Somatic Cell Hybrid Mapping

This unit provides an introduction to the various types of hybrid panels in use and reviews the status of commercially available panels.

Complete cDNA encoding human phospholipid transfer protein from human endothelial cells.

Phospholipid transfer protein, with an apparent molecular mass of 81 kDa, was purified from human plasma. The NH2-terminal amino acid sequence of a 51-kDa proteolytic fragment obtained from phospholipid transfer protein allowed degenerate primers to be designed for polymerase chain reaction and the eventual isolation of a full-length cDNA from a human endothelial cDNA library. The cDNA is 1,750 base pairs in length and contains an open reading frame of 1,518 nucleotides encoding a leader of 17 amino acids and a mature protein of 476 residues. Northern blot analysis shows a single mRNA transcript of approximately 1.8 kilobases with a wide tissue distribution. The gene was mapped to chromosome 20 using a human/rodent somatic cell hybrid mapping panel. Phospholipid transfer protein was found to be homologous to human cholesteryl ester transfer protein, human lipopolysaccharide-binding protein, and human neutrophil bactericidal permeability increasing protein (20, 24, and 26% identity, respectively).

Primate testicular histone H1t genes are highly conserved and the human H1t gene is located on chromosome 6.

The testis-specific histone H1t gene is known to be transcribed only in pachytene primary spermatocytes during spermatogenesis. Previous studies of the rat histone H1t gene revealed a unique promoter sequence element between the H1/GC box and the H1/CCAAT box. Proteins in crude nuclear extracts of rat testis bind specifically to this sequence element and a temporal correlation exists between the appearance of these DNA binding proteins and the onset of transcription. These discoveries led to a search for histone H1t genes in other mammalian species. The human and monkey histone H1t genes were amplified from genomic DNA using the polymerase chain reaction (PCR). The amplified genes were cloned and the genomic derived inserts were sequenced using linear PCR. Both proximal promoters contained the highly conserved H1/AC box, H1/CCAAT box, and H1/TATA box found in nongerminal H1 genes. Both promoters also contained the H1/GC box and the H1t/CCTAGG sequence element between the H1/GC box and H1/CCAAT box previously seen only in the H1t promoter. Specific amplification of the human H1t gene using template DNA samples from a NIGMS human/rodent somatic cell hybrid mapping panel has shown that the human histone H1t gene is located on chromosome 6.

Physical Mapping of the NF2/Meningioma Region on Human Chromosome 22q12

Loss of genetic information from chromosome 22 has been implicated in the development of neurofibromatosis type 2, meningioma, and several other neoplasia. Molecular studies indicate that genes within chromosomal band 22q12 may be involved in tumorigenesis. We have mapped 29 loci into 16 groups in this region, using pulsed-field gel electrophoresis, fluorescence in situ suppression hybridization, and somatic cell hybrid mapping. The region spans more than 5 Mb of genomic DNA and contains the genes for neurofibromatosis type 2 and meningioma. The order of loci presented here provides the framework for the fine mapping of this region using cosmids and yeast artificial chromosomes, and it facilitates the speedy cloning of novel genes from 22q12.

Confirmation of the Assignment of the Human Tartrate-Resistant Acid Phosphatase Gene (ACP5) to Chromosome 19

Tartrate-resistant acid phosphatase (or type 5 acid phosphatase; EC 3.1.3.2) is an iron-binding protein localized primarily in osteoclasts that may play an important role in the bone resorption process. The gene has previously been localized in the human genome by two different groups. Allen et al. localized the gene to human chromosome 15 (15q22-q26) using the technique of in situ hybridization, while Lord et al. localized the gene to human chromosome 19 (19p13.2-13.3) using both in situ hybridization and a somatic cell hybrid mapping panel. 6 refs., 1 fig.

Assignment of a human voltage-dependent sodium channel alpha-subunit gene (SCN6A) to 2q21-q23.

Voltage-dependent sodium (Na[sup +]) channels are responsible for the initial membrane depolarization that occurs during action potential generation in most electrically excitable cells. These ion channel proteins exist as heteromultimeric complexes of a large ([approximately]260 kDa) [alpha]-subunit and one or two smaller ([approximately]33-38 kDa) [beta]-subunits. A multigene family consisting of at least five structurally related Na[sup +] channel [alpha]-subunit isoforms has been defined. Three brain Na[sup +] channel isoform genes are clustered together on proximal mouse chromosome 2 and compose part of a linkage group that exhibits synteny with a region of human 2q. We now have localized the gene encoding the hNa 2.1 Na[sup +] channel isoform to this same general region of chromosome 2 (2q21-q23) by a combination somatic cell hybrid mapping and chromosome microdissection-polymerase chain reaction. This information may facilitate the identification of inherited diseases affecting Na[sup +] channel function. 10 refs., 1 tab.

Chromosome assignment of human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels

Sixty-three human brain cDNA sequences were newly assigned to individual human chromosomes. Ten of these were subregionally localized, and one was also mapped in the mouse genome. Four previously reported assignments were refined. PCR primers were designed from expressed sequence tags (ESTs) and tested for specific amplification from human genomic DNA. DNA was then amplified, often in multiplexed PCR reactions, using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. Thirteen newly localized cDNAs exhibited homology to previously reported sequences. EST01471 was shown to correspond to human microtubule-associated protein 1B (MAP1B), confirming the previous mapping of this gene to human chromosome 5. Other genes tentatively assigned to chromosomes based on these results were a component of the signal peptide receptor of the endoplasmic reticulum (EST00745) and a cyclic AMP-regulated phosphoprotein (EST01041) on chromosome 1, a protein phosphatase 2A 55-kDa regulatory subunit (EST01650) on chromosome 4, an NAD(P) transhydrogenase (EST01744) on chromosome 5, ribosomal proteins L1a or L1b (EST01627) and L18a (EST01583), a brain transcription factor (BF-1, EST00795) on chromosome 14, a milk fat globule membrane-related protein (EST01678) on chromosome 15, a putative peptide initiation factor (EST00675) on chromosome 17, thiosulfate sulfurtransferase (TST) on chromosome 22, and moesin (MSN) (EST00896) and a human equivalent of rat spot 14 (S14) (EST00887) on Xp11-->cen and Xpter-->p21.3, respectively.

Assignment of the gene for the core protein II (UQCRC2) subunit of the mitochondrial cytochrome bc1 complex to human chromosome 16p12.

The mammalian cytochrome be[sub 1] complex (complex III) of the mitochondrial respiratory chain catalyzes electron transfer from ubiquinol to cytochrome c. The complex consists of 10-11 subunits: Core proteins I and II, cytochromes b and c[sub 1], the Rieske iron-sulfur protein, the ubiquinone-binding protein, the hinge protein, and 3-4 subunits of low molecular weight. Cytochrome b is encoded by the mitochondrial genome; the other subunits are encoded by nuclear genes. Both the human cytochrome c[sub 1] and the human ubiquinone-binding protein subunits have been assigned to chromosome 8 by somatic cell hybrid mapping. In this study, the authors used in situ hybridization to map core protein II. In situ hybridization to BrdU-synchronized peripheral blood lymphocytes was performed using the method of Harper and Saunders. Chromosomes were stained with a modified fluorescence, 0.25% Wright's stain procedure. The positions of silver grains directly over or touching well-banded metaphase chromosomes were mapped to an ISCN idiogram. The analysis of the distribution of 200 silver grams following in situ hybridization revealed a significant clustering of grains in the p12 region of chromosome 16. The assignment of the core II subunit to human chromosome 16p12 confirms that it is encoded by the nuclear, rather thanmore » the mitochondrial, genome. The identification of a single strong hybridization signal is indicative of one locus with no pseudogenes. 6 refs., 1 fig.« less

Assignment of four sequence-tagged sites to three subregions of 13q12 using a somatic cell hybrid mapping panel.

To define the position of a 13q12 breakpoint from a patient with ganglioneuroblastoma, a series of somatic cell hybrids carrying human chromosome translocations with breakpoints in the proximal part of chromosome 13 has been compiled. Sequence-tagged sites (STS) have been generated from a series of Alu-PCR probes previously shown to be in the 13q12 region. Together with an STS for the oncogene FLT1, these have been used to define the relative positions of the translocation breakpoints in the hybrids. In this way, four markers have been ordered in three subregions of 13q12 and reference breakpoints established. The refined physical map of 13q12 provides a series of reference markers with known locations and will be invaluable in the further characterization of breakpoints in this region.

Assignment of the Human Pulmonary Surfactant Protein D Gene (SFTP4) to 10q22-q23 Close to the Surfactant Protein A Gene Cluster

Pulmonary surfactant consists of a complex mixture of phospholipids and several proteins essential to normal respiratory function. Two of the surfactant proteins, SP-A and SP-D, appear to have lectin-like activity relevant to the local phagocytic defense. Using polymerase chain reaction (PCR)-based somatic cell hybrid mapping, the human SP-D gene (SFTP4) was assigned to chromosome 10. A regional mapping panel was assembled and characterized using sequence tagged sites for five loci previously mapped to 10q. SFTP4, the SP-A gene (SFTP1), and the microsatellite D10S109 were placed in the interval 10q22-q23. Low-stringency PCR using the SFTP1 primer pair suggested the presence of at least two additional SP-A-related genes in the same region. With the locus for mannose-binding lectin (MBL) at 10q21, this may be indicative of this region's central role in the evolutionary history of carbohydrate-binding proteins containing collagen-like regions.

NIGMS human/rodent somatic cell hybrid mapping panels 1 and 2.

The NIGMS Human Genetic Mutant Cell Repository is currently distributing two well-characterized human/rodent somatic cell hybrid mapping panels. Mapping Panel 1 consists of DNA isolated from 18 hybrid cell cultures retaining from 1 to 19 human chromosomes. Mapping Panel 2 contains DNA from hybrids retaining 1 or 2 human chromosomes. All but 2 of the hybrids retain a single intact human chromosome. Mapping Panel 2 is also available as cell cultures. These resources should prove valuable to the Human Genome Project initiative. This article describes the sources of the hybrid cell cultures and the procedures utilized to prepare and characterize the panels.

Characterization of NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2 by PCR

NIGMS human somatic cell hybrid mapping panel 2 was tested for specific markers for each human chromosome. Several discrepancies with the reported chromosome content were found by screening with PCR primers. Our results indicated that there were additional chromosomal fragments present in some of the cell lines and that a few of the hybrids had lost part of the reported chromosome.

Transcriptional regulation of TSG6, a tumor necrosis factor- and interleukin-1-inducible primary response gene coding for a secreted hyaluronan-binding protein.

TSG6 was originally identified as a tumor necrosis factor (TNF)-inducible gene in human fibroblasts. Earlier we showed that the secretory TSG6 protein is a member of a family of hyaluronan-binding proteins that includes cartilage link protein, proteoglycan core protein, and the adhesion receptor CD44. In the present study we have used Southern blot analysis to demonstrate that TSG6 is a single-copy gene in the human and murine species. With the aid of a somatic cell hybrid mapping panel, TSG6 was assigned to human chromosome 2. Nuclear run-on analysis revealed that TNF produced a rapid, primary transcriptional activation of the TSG6 gene in normal human FS-4 fibroblasts. In order to learn more about the regulation of TSG6 gene expression, we cloned the TSG6 gene from a genomic library of human white blood cells. Sequencing of a 1.3-kilobase fragment of the 5'-flanking region of the TSG6 gene identified TATA-like and CAAT sequences near the transcription start site. In addition, potential binding sites for NF-IL-6, AP-1, interferon regulatory factors (IRF)-1 and -2, and glucocorticoid response elements were identified in the 5'-flanking region. A single transcription start site was identified by primer extension. Deletion analysis of the 5'-flanking region of the TSG6 DNA linked to the chloramphenicol acetyltransferase reporter gene revealed that a construct containing TSG6 DNA from positions -165 to +78 could be transcriptionally activated by interleukin(IL)-1, and to a lesser extent by TNF, upon transfection into FS-4 fibroblasts. The region that imparts inducibility by IL-1 or TNF (positions -165 to -58) contains potential binding sites for IRF-1 and -2, AP-1, and NF-IL-6. A region mediating transcriptional silencing was localized further upstream (between positions -332 and -165). The results suggest that TSG6 gene expression is regulated by an interplay of positively and negatively acting transactivating factors.

Physical and Genetic Mapping of a Human Apical Epithelial Na+/H+ Exchanger (NHE3) Isoform to Chromosome 5p15.3

A gene family of Na+/H+ exchanger isoforms has been identified. Characterization of rabbit NHE3 suggests that it is the apical epithelial Na+/H+ exchanger isoform responsible for transepithelial, electroneutral Na+ absorption in intestinal and renal epithelial cells. We have previously isolated from a human kidney cortex library a partial human NHE3 cDNA, clone HKC-3. Using HKC-3 to probe human/rodent somatic cell hybrid mapping panels, the human NHE3 gene was physically mapped to the distal portion of chromosome 5p15.3. Southern analysis of EcoRI-digested human genomic DNAs of CEPH pedigrees probed with HKC-3 detected three polymorphic sites containing a total of nine alleles that segregate in a Mendelian fashion. The observed heterozygosity for the NHE3 locus in unrelated individuals was 71%. Linkage analysis between NHE3 and other markers known to map at 5p15 confirmed the localization of NHE3 to chromosome 5p15.3, making NHE3 the most telomeric gene identified on the short arm of chromosome 5.

Somatic Cell Hybrid Mapping on Mouse Chromosome 11 (MMU11): Assignment of Markers Relative to Two Breakpoints in Band D

Mouse × rat somatic cell hybrids were generated by fusing mouse cell lines that are heterozygous for reciprocal translocations involving the T42H and T9Ad breakpoints on mouse chromosome 11 (MMU11) to a thymidine kinase-negative (Tk -) rat cell line, RT 2Tk -. Selection in HAT medium with geneticin disulfate (G418) resulted in some hybrid clones retaining only one derivative translocation chromosome with that part of MMU11 carrying the Tk-1 locus. Southern blot and PCR analyses of these hybrids were used to map the two breakpoints and 30 markers relative to them. The T42H breakpoint has been localized between Mpo and the Cola-1/Hox-2 cluster of loci and is proximal to the T9Ad breakpoint. The T9Ad breakpoint is proximal to the distal loci Tk-1, Gaa, D11Jkn1, and P4hb . The positions of 14 loci ( Hox-2, Cola-1, Rara, Phb, Erba, Rnula-1, D11Pas1, Gfap, D11Mit13, D11Mit11, D11Mit12, Myla, Empb3 and Gh ) have been further refined by their localization between the two breakpoints in band D. This study therefore improves the correlation of the genetic and physical maps of MMU11 and extends the known homology between MMU11 and human chromosome 17 (HSA17) by the assignment of three additional HSA17 markers, the profilin gene, Pfn, an anonymous marker, D17s28h, and the Crk oncogene, to above the T42H breakpoint; and the prohibitin gene, Phb, to between the T42H and T9Ad breakpoints in band D on MMU11.

Glycosylation-dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterization of two highly similar enzyme forms.

A cDNA, designated hCE, encoding the entire sequence of a carboxylesterase, was isolated from a human liver lambda gt11 library. The hCE-deduced protein sequence contained 568 amino acids, including an 18 amino acid signal peptide sequence, and had a calculated molecular mass of the mature protein of 60,609 Da. A second cDNA, designated hCEv, was isolated from the same lambda gt11 library and contained a 3-bp deletion resulting in the loss of the final amino acid in the signal peptide sequence (Ala-1) and a second 3-bp deletion leading to an in-frame loss of Gln345. Expression of mRNA corresponding to both hCE and hCEv was detected in eight adult human liver samples, with individual levels varying 5-fold (hCE) and 12-fold (hCEv). A single immunoreactive protein was detected in 13 adult human liver samples when probed with antibody directed against a rat carboxylesterase. Based on allele-specific oligonucleotide hybridizations, we believe that the hCE and hCEv cDNAs represent two distinct members of the carboxylesterase family. The carboxylesterase genes were localized to human chromosome 16 using a somatic cell hybrid mapping strategy. Baculovirus expression of hCE in Sf9 cells produced a protein with an estimated molecular mass of 59,000 Da. This enzyme was able to hydrolyze aromatic and aliphatic esters but possessed no catalytic activity toward amides or a fatty acyl CoA ester. Baculovirus-mediated expression of the hCEv cDNA yielded a second protein of 56,000 Da resulting from inefficient N-glycosylation of the hCEv protein. Although the substrate specificity for the hCEv protein was identical to that of expressed hCE for any given substrate, the specific activity for the hCE protein was always higher than that for the hCEv protein. Tunicamycin inhibition studies provided the first evidence that N-glycosylation of these luminal enzymes is essential for maximal catalytic activity.

Isolation of cosmids and fetal brain cDNAs from the proximal long arm of human chromosome 22.

The proximal portion of human chromosome 22q appears to carry genes implicated in the pathogenesis of various developmental disorders, including the cat eye syndrome (CES) and the DiGeorge syndrome (DGS). A cosmid library was prepared from a radiation hybrid selected for its content in chromosome 22 fragments. A large fraction of cosmids containing human DNA were found to derive from the juxtacentromeric region of chromosome 22, as shown by fluorescence in situ hybridization (FISH) performed using individual cosmids or cosmid pools as probes. Finer mapping was obtained for individual cosmids by hybridization to a somatic cell hybrid mapping panel which splits the long arm of the chromosome into 14 bins numbered 1 to 14 from the centromere to the telomere. Of the 10 cosmids mapped, eight belonged to group 1, the other two to group 14, in agreement with FISH data. Rare endonuclease sites and fragments conserved between species were searched in single cosmids, resulting in the selection of seven cosmid fragments which were used to screen a human fetal brain cDNA library. Three cDNAs were identified, encoded from two chromosome 22 genes which appeared to be novel, as determined from partial end sequence and comparison with the database entries. Fine localization of the 30.9 cDNA indicated that the corresponding gene was located in a segment of proximal 22q overlapping with the critical DGS region.

Human connexin43 gene locus, GJA1, sublocalized to band 6q21-->q23.2.

Gap junctions permit transfer of small molecules directly between cells. Several laboratories have mapped the human gap junction protein subunit, connexin43 (locus designation, GJA1), to overlapping regions of chromosome 6, localizing GJA1 to 6q14-->q24. We have sublocalized human GJA1 to a very small region. 6q21-->q23.2, using a human x rodent somatic cell hybrid mapping panel and a rat connexin43 cDNA probe.

Mapping of the human adenylosuccinate lyase (ADSL) gene to chromosome 22q13.1-->q13.2.

Adenylosuccinate lyase (ADSL) is an essential enzyme involved in de novo purine biosynthesis. A deficiency in ADSL in humans has been shown to predispose to a neurodevelopmental syndrome with autistic features. Using both a somatic cell hybrid mapping panel and fluorescence in situ hybridization, we have precisely localized the human ADSL gene to chromosome 22q13.1-->q13.2.