Structure and chromosomal location of the rat ribophorin I gene.

Ribophorin I is a type I transmembrane glycoprotein characteristic of the rough portions of the endoplasmic reticulum where it is thought to play a role in the cotranslational insertion of nascent polypeptides. A rat ribophorin I cDNA was used to isolate four overlapping genomic clones from a rat EMBL3 genomic library. Restriction mapping, Southern blotting, and DNA sequencing showed that these clones, spanning approximately 21 kilobases of chromosomal DNA, include the entire ribophorin I gene, as well as 15 kilobases (kb) of upstream sequences. Southern blotting analysis of DNA from a panel of mouse-Chinese hamster cell hybrids demonstrated that the ribophorin I gene is located on mouse chromosome six. The ribophorin I gene contains 10 exons, seven of which encode the luminal domain of the polypeptide. Exon 8 encodes the trans-membrane domain and small portions of the flanking luminal and cytoplasmic domains. Exons 9 and 10 encode the remainder of the cytoplasmic domain, and the latter includes the 3'-untranslated portion of the mRNA. Six closely spaced transcription start sites located 3 to 24 base pairs upstream from the initiation codon were identified by primer extension analysis and S1 mapping. The sequence of a 1.3-kb region upstream of the cap sites was determined and found to contain three GC-rich potential Sp1-binding sites beginning at -14, -24, and -91 base pairs (bp), two octamer-like sequences at -233 and -1248 bp, and a CAAT-like box at -41 bp. The possible roles of these elements in regulating expression of the ribophorin gene in all cells and in differentiated cell types characterized by a well developed rough endoplasmic reticulum is discussed.

Attenuation of opioid receptor activity by phorbol esters in neuroblastoma x glioma NG108-15 hybrid cells.

Chronic opioid treatment of neuroblastoma x glioma NG108-15 cells induces desensitization of the opioid receptor and this may involve a change in membrane protein phosphorylation. In an attempt to mimic this possible mechanism, we studied effects of phorbol ester activation of protein kinase C on opioid receptor activity. Incubation of NG108-15 hybrid cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) abolished up to 45% of opioid inhibition of cyclic AMP accumulation in intact cells, while basal accumulation and prostaglandin E1-stimulated cyclic AMP accumulation were unaltered. This decrease of opioid inhibition was dose- and time-dependent and the potency order of phorbol esters and apparent K activation (90 nM) for TPA were consistent with phorbol esters acting through the stimulation of protein kinase C. TPA also decreased the inhibition of cyclic AMP accumulation mediated through muscarinic and alpha-2 adrenergic receptors. These effects of TPA were best explained by a TPA-induced alteration of the inhibitory nucleotide-binding protein (Gi), the common transducer protein of these receptors. Impairment of Gi by TPA treatment was evidenced by a reduction in agonist-stimulated GTP hydrolysis and activation by GTP. Quantification of Gi by pertussis toxin-catalyzed ADP-ribosylation revealed that TPA decreased maximal labeling. In summary, phorbol esters appeared to attenuate opioid receptor activity by altering the activity of the transducer protein Gi.

The gene for the beta-subunit of retinal transducin (Gnb-1) maps to distal mouse chromosome 4, and related sequences map to mouse chromosomes 5 and 8.

The heterotrimeric G protein transducin releases cGMP-phosphodiesterase from inhibition in retinal rod photoreceptor cells when stimulated by light-activated rhodopsin. As a result the level of cGMP goes down, the rod plasma membrane hyperpolarizes, and the release of neurotransmitter is modified. We have used a bovine cDNA for the beta-subunit of transducin (G beta 1) to map its gene Gnb-1 to distal mouse chromosome 4. This cDNA also identified two other homologous sequences in the mouse genome. One of the sequences was on chromosome 5 which we identified as the locus of Gnb-2, a second G protein beta-subunit gene. The other sequence was on chromosome 8 and is either a pseudogene or an as yet undiscovered third G beta-subunit gene, here termed Gnb-3.

Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequence polymerase chain reaction.

A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3' end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300-1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromide-stained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.

Long-range physical mapping around the human steroid sulfatase locus.

The region of the human X chromosome containing the steroid sulfatase locus was analyzed by pulsed-field gel electrophoresis. Restriction site maps were generated for the X chromosome in the blood of a normal male individual and that in the mouse-human hybrid cell line ThyB-X; these maps extend over approximately 4.3 Mb of DNA of the former, and 3.2 Mb of the latter. Physical linkage was defined between the STS locus and sequences detected by the probes GMGX9 (DXS237), GMGXY19 (DYS74), CRI-S232 (DXS278), and dic56 (DXS143), and the order telomere--(STS, DYS74)--DXS237--DXS278--DXS143--centromere was deduced. The pulsed-field maps were used to demonstrate a deletion of 180 kb of DNA from the X chromosome of an individual with X-linked ichthyosis. Also, possible locations for the Kallmann syndrome gene were revealed, and the distance between the steroid sulfatase locus and the pseudoautosomal region was estimated to be at least 4 Mb.

The gene for the RNA component of the mitochondrial RNA-processing endoribonuclease is located on human chromosome 9p and on mouse chromosome 4.

Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5' flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHM1.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21--p12.

Characterization and localization to human chromosome 1 of human fast-twitch skeletal muscle calsequestrin gene.

A genomic clone encoding human fast-twitch skeletal muscle calsequestrin was isolated, and the amino acid sequence of the protein and the exon-intron boundaries of the gene were deduced from its sequence. A comparison with the rabbit gene showed that the sequence Glu-Asp-Asp-Asp-Asp near the COOH terminus of the rabbit sequence is lacking in the human gene. The calsequestrin gene was assigned to human chromosome 1 through the use of a human-mouse somatic cell hybrid mapping panel.

Absence of uvomorulin in a slowly compacting variant of H6 embryonal carcinoma cells.

When allowed to aggregate in calcium-containing medium, the H6 embryonal carcinoma cell variant named 6B(NG)C25 compacted more slowly than wild-type cells, and aggregates of hybrids between it and wild-type cells also compacted slowly, as if the variation (mutation) acted in a dominant fashion. In agreement with this, we now have found that the cell adhesion molecule uvomorulin is markedly reduced or absent in 6B(NG)C25 cells, as well as in the hybrids. A small amount of a higher-molecular-weight protein reacting with the antibody is present, which might represent residual uvomorulin migrating at a slower rate, an altered uvomorulin, the known precursor to uvomorulin, or an unrelated cross-reacting protein.

Intragenic interspecific complementation of glucose 6-phosphate dehydrogenase in human-hamster cell hybrids.

A new variant of human glucose 6-phosphate dehydrogenase (G6PD), provisionally designated G6PD Harilaou, was observed in a Greek boy affected by severe hemolytic anemia. G6PD Harilaou was associated with very severe deficiency of enzyme activity, which measured about 1% of normal in the patient's fibroblasts. By fusion of Harilaou fibroblasts with a similarly enzyme-deficient mutant CHO cell line, we have isolated a hybrid cell line that has a G6PD activity 5-10 times higher than either of the parental cells. By electrophoretic analysis we show that most of this activity is associated with a hybrid dimeric G6PD molecule consisting of one hamster and one human subunit. We suggest that this heterologous quasi-interallelic complementation is effected by a catalytically abnormal hamster subunit stabilizing a catalytically abnormal and unstable Harilaou subunit. This approach may be useful for the study of dimer formation and stability in human G6PD.

Mapping of the L-methylmalonyl-CoA mutase gene to mouse chromosome 17.

In humans, methylmalonyl acidemia is caused by a deficiency of L-methylmalonyl-CoA mutase (MUT) controlled by a gene that has been mapped to chromosome 6. The mouse homolog of this gene has now been mapped to mouse chromosome 17. Recombinant inbred and congenic strains place the mouse Mut locus 1.06 cM distal to H-2, between Pgk-2 and Ce-2. The relative order of syntenic probes flanking H-2 on mouse chromosome 17 and HLA on human chromosome 6 is shown to be different.

Completion of the primary structure of the human type IV collagenase preproenzyme and assignment of the gene (CLG4) to the q21 region of chromosome 16.

The complete amino acid sequence of the human type IV collagenase preproenzyme was determined from cDNA and genomic clones. Primer extension and S1 nuclease analyses as well as nucleotide sequencing of a genomic clone indicate that the first exon has two closely spaced initiation sites for transcription and codes for 290 and 280 nt of a 5' untranslated region and a 29-residue signal peptide. The gene (CLG4) was localized to 16q21 using somatic cell hybrids and in situ hybridization.

Generation of a panel of somatic cell hybrids containing unselected fragments of human chromosome 10 by X-ray irradiation and cell fusion: application to isolating the MEN2A region in hybrid cells.

We have used X-ray irradiation and cell fusion to generate somatic cell hybrids containing fragments of human chromosome 10. Our experiments were directed towards isolating the region of the MEN2A gene in hybrids and to use those as the source of DNA for cloning and mapping new markers from near the MEN2A locus. A number of hybrid clones containing human sequences that are tightly linked to the MEN2A gene were identified. Some 25% of our hybrids, however, proved to contain more than one human chromosome 10-derived fragment or showed evidence of deletions and/or rearrangements. A detailed analysis of the human content of X-ray irradiation hybrids is required to assess the integrity and number of human fragments retained. Despite retention of multiple human-derived fragments, these hybrids will prove useful as cloning and mapping resources.

Microdissection and microcloning of human chromosome 7q22-32 region.

Genetic information contributing to cystic fibrosis in addition to the CF gene is suggested to reside on the long arm of the human chromosome 7. In our attempt to analyze this genomic region in detail, we generated a region-specific DNA probe library by microdissection and microcloning of the midpiece of the chromosome 7q arm. Microdissection was performed in unstained metaphase spreads from a human x mouse hybrid cell line containing chromosome 7 as the only human chromosome. We obtained 593 clones from 75 dissected chromosomal fragments. At least 88% of the microclones were true recombinants; 40% of the clones contained repetitive sequences as determined by plaque hybridization with genomic DNA as probe. The overall mean fragment size of insert fragments was 3.2 kb, the median size was 3.5 kb. Regional mapping of 30 DNA fragments was performed by the aid of hybrid cell lines containing different segments of human chromosome 7; 50% of the microcloned inserts were found to map to 7q22-32.

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

Chromosomal assignment of the gene for the ubiquinone-binding protein of human mitochondrial cytochrome bc1 complex.

The ubiquinone-binding protein (QP-C) is a nuclear-encoded subunit of the cytochrome bc1 complex in the mitochondrial respiratory chain and is thought to be involved in the electron transfer reaction coupled to energy transduction. We recently isolated a nuclear gene for human QP-C and used, in the present study, its fragment as a probe for Southern blot analysis of EcoRI-digested DNAs prepared from 14 human-mouse somatic cell hybrids. The results indicated that the human QP-C gene is located on chromosome 8.

Analysis of somatic cell hybrids derived from normal human prostatic epithelial cells fused with HeLa cells.

Somatic cell hybrids have been instrumental in the recognition of specific chromosomes containing genes capable of suppressing the malignant phenotype. As a first step towards the identification of possible suppressor genes in prostate cells, we created hybrids by fusing normal prostate cells with malignant HeLa cells. Similar to hybrids made with other combinations of normal and malignant cells, the normal phenotype was dominant and the malignant phenotype was suppressed. The phenotype of the nontumorigenic hybrids after injection into nude mice resembled that of normal keratinocyte X HeLa hybrids, and tiny, nonprogressive keratinized nodules were produced. One hybrid clone was tumorigenic, possible due to the loss of a normal suppressor gene, and displayed glandular as well as squamous elements. Further characterization of these hybrids should permit isolation of specific suppressor genes, as well as promote recognition of elements that regulate the glandular phenotype.

Absence of Ig V region gene somatic hypermutation in advanced Burkitt's lymphoma.

Somatic hypermutation of rearranged Ig V region gene plays a major role in generating antibody diversity. Recently, V mutation has been established as a major mechanism of tumor escape from anti-Id immunotherapy. We cloned and sequenced the expressed Ig H and L chain V regions from a case of B acute lymphoblastic leukemia in order to evaluate B cell stages associated with V region mutation, and to determine which tumors would be better suited to Id directed immunotherapy. A consensus VH and V lambda sequence representing tumor at diagnosis was obtained by conventional cDNA cloning in lambda gt10 from a heterohybridoma. Primers which flanked both V regions were used in a modified polymerase chain reaction to generate multiple independent sequences from tumor cells harvested at relapse. In order to exclude mutations due to infidelity of the amplification procedure, single cDNA templates of known sequence were also amplified. The polymerase chain reaction proved to be an effective procedure to obtain multiple clones, but replication in M13 was associated with a low rate of base misincorporation. The results indicate that there is no evidence for biologically significant ongoing mutation in this t(8;14) B cell tumor when comparing sequences at diagnosis and relapse. Thus, V somatic mutation may be restricted to a discrete B cell stage whose malignant counterpart is follicular lymphoma.

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation.

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.

Saturation of human chromosome 3 with unique sequence hybridization probes.

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.

Genetic variation in an inbred plant: variation in tissue cultures of soybean [Glycine max (L.) Merrill].

Although soybean [Glycine max (L.) Merrill] grows as an inbreeding, generally homozygous, plant, the germplasm of the species contains large amounts of genetic variation. Analysis of soybean DNA has indicated that variation of RFLP (restriction fragment length polymorphism) markers within the species usually entails only two alleles at any one locus and that mixtures of such dimorphic loci account for virtually all of the restriction fragment variation seen in soybean (G. max), and in its ancestors, G. soja and G. gracilis. We report here that tissue cultures prepared from root tissue of individual soybean plants develop RFLP allelic differences at various loci. However, these newly generated alleles are almost always the same as ones previously found and characterized in other varieties of cultivated soybean (cultivars). This repeated generation of particular alleles suggests that much of the genetic variation seen in soybean could be the consequence of specific, relatively frequently employed, recombinational events. Such a mechanism would allow inbred cultivars to generate genetic variation (in the form of alternative alleles) in a controlled manner, perhaps in response to stress.