Synteny between the pro+ marker and human glutamate oxaloacetate transaminase.

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.     

Localization of a gene which complements branched-chain amino acid transaminase deficiency to the short arm of human chromosome 12

Summary A humanxChinese hamster somatic cell hybrid (J1) containing only one human chromosome (number 11) does not express branched-chain amino acid transaminase and, therefore, cannot grow if the branched--keto acids are substituted for the branched-chain amino acids in the growth medium. J1 cells, which are glycine auxotrophs (Gly A), were fused with normal human lymphocytes and the resulting hybrids selectively isolated in glycine-free medium. A total of 16 primary and 46 secondary clones were analyzed for isozymic and nutritional markers. Cytogenetic analysis with chromosome banding was also performed on selected hybrid clones. The results provide evidence that branched-chain amino acid transaminase is syntenic with lactic dehydrogenase B and is located on the short arm of human chromosome 12.     

Gene order on the short arm of human chromosome 11: regional assignment of the LDH A gene distal to catalase in two translocations

Summary Leukemic cells with reciprocal translocations involving 11p13 and 14q13 were obtained from two patients with T-cell acute lymphoblastic leukemia and fused with mouse Ltk^- cells. DNA from independent hybrid clones was screened by Southern blot and hybridization to molecular probes for the human catalase and Ha-ras-1 genes. Several clones showed segregation of these two genes, indicating the presence of either the der 11 or der 14 human chromosomes. When DNA from these hybrid clones was examined for the presence of the human genes for calcitonin and γ-globin, both genes were found to segregate with the Ha-ras-1 gene and the der14 chromosome indicating that they lie distal to catalase. When the hybrid clones were examined for the presence of human lactate dehydrogenase A (LDH A) activity, only those clones containing the der14 chromosome expressed activity indicating that the LDH A gene is also distal to catalase on the short arm of chromosome 11.     

Cotransfer and phenotypic stabilisation of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer

Summary By means of metaphase chromosomes, the genes for mink thymidine kinase (TK) and hypoxanthine-phosphoribosyltransferase (HPRT) were transferred to mutant mouse cells, LMTK^-, A9 (HPRT^-) and teratocarcinoma cells, PCC4-aza 1 (HPRT^-). Eighteen colonies were isolated from LMTK^- (series A), 9 from A9 (series B) and none from PCC4-aza 1. The transformed clones contained mink TK or HPRT. Analysis of syntenic markers in series B demonstrated that one clone contained mink glucose-6-phosphate dehydrogenase (G6PD) and the other alpha-galactosidase; in series A, nine clones contained mink galactokinase (GALK) and six mink aldolase C (ALDC). Analysis of 12 asyntenic markers located in ten mink chromosomes showed the presence of only aconitase-1 (ACON1) (the marker of mink chromosome 12) in three clones of series A. The clones lost mink ACON1 between the fifth to tenth passages. Cytogenetic analysis established the presence of a fragment of mink chromosome 8 in eight clones of series A, but not in series B. The clones of series A lost mink TK together with mink GALK and ALDC during back-selection; in B, back-selection retained mink G6PD. No stable TK⁺ phenotype was detected in clones with a visible fragment of mink chromosome 8. Stability analysis demonstrated that about half of the clones of series B have stable HPRT⁺ phenotype whereas only three clones of series A have stable TK⁺ phenotype. It is suggested that the recipient cells, LMTK^- and A9, differ in their competence for genetic transformation and integration of foreign genes.     

Studies on phenotypic variation between Chinese hamster Kupffer cell lines : II. Correlation relationships and coordinate control of enzyme activities

This study examines somatic cell hybrids between parental cells of identical origin which exhibited quantitative differences in enzyme activities. Nine enzymes in cultured Chinese hamster Kupffer cell hybrids were studied. The parental Kupffer cell clones of identical genetic origin differed several-fold in the specific activities of catalase, arginase, microsomal heme oxygenase, peroxidase, β-glucuronidase, alcohol dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. A selective system based on fusion of ouabain and vinblastine resistant clones of the parental cells was used to isolate hybrids. In hybrid clones, the specific activities of catalase, microsomal heme oxygenase, peroxidase and the dehydrogenases were expressed at the level characteristic of the parental clone with high activities of these enzymes. This result implied interaction between the parental genomes and suggested mechanisms regulating the quantitative levels of several enzyme activities. In contrast, the specific activities of arginase and β-glucuronidase in hybrid clones were intermediate between those possessed by the parental cells and indicated that for each parental genome in the hybrid there was autonomous regulation of the levels of these two enzyme activities.     

Separation of Linked Markers in Chinese Hamster Cell Hybrids: Mitotic Recombination Is Not Involved

A search for mitotic recombination was carried out using mutant subclones of cultured Chinese hamster ovary cells. Recombination events were sought between the linked loci specifying the enzymes hypoxanthine phosphoribosyl transferase and glucose-6-phosphate dehydrogenase. It was shown by fluctuation analysis that markers at these two loci co-segregate from doubly heterozygous pseudotetraploid hybrid cells more than 90% of the time. The minority class of segregants, which had lost one marker without losing the other, were genetically analyzed to distinguish between the possibilities of mitotic recombination and deletion of chromosomal material. Nine clones in which a linkage disruption had occured were studied, using further cell hybridization and segregation. In three cases, a recessive lethal loss of genetic information was indicated, suggesting the deletion mechanism. In six cases, it was demonstrated that no new linkage relationships had been established concomitant with linkage disruption. Thus, in all nine clones, the evidence indicated that mitotic recombination was not involved in the events that disrupted linkage between these two loci. If mitotic recombination takes place at all in this system, the rate must be less than about 10^-6 per cell per generation.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

Human cell surface antigens coded by genes on chromosome 21

Clones of man-mouse hybrids derived from four different crosses which retained a very limited number of human chromosomes were studied for the expression of human cell surface antigens. In testing a variety of rabbit antisera to human cells and tissues, it was found that an antiserum to Daudi cells recognizes human species-specific antigen(s) on three ‘WA’ clones, all of which carried human chromosome 21. Absorption of the antiserum with any of the clones abolished its activity against all clones, indicating that the antiserum recognized the same antigen(s) on these clones. The antigen(s) was shown to be present on normal human lymphocytes, more on B than on T cells, but apparently absent from erythrocytes. C3H mice, from which the murine parent originated, were immunized with the WA clones carrying human chromosome 21. The resultant antisera reacted with clones carrying chromosome 21 but not with clones which did not retain this chromosome, even though some of these clones possessed many other human chromosomes. The murine antisera reacted with some, but not all, human peripheral blood lymphocytes tested. Absorption studies clearly showed the multiple nature of the antigens recognized by these antisera. Studies on cells of identical twins provided evidence that these antigens are inheritable.     

Assignment of the gene for lactic dehydrogenase A to mouse chromosome 7 using mouse-human hybrids

We have studied somatic cell hybrids between either mouse peritoneal macrophages or spleen cells and HT-1080-6TG human fibrosarcoma cells for the expression of mouse lactic dehydrogenase A (LDH-A). The hybrids were also studied for the expression of mouse glucose phosphate isomerase (GPI-1), the gene for which has been assigned to chromosome 7. Concordant segregation of the expression of mouse GPI-1 and LDH-1 was observed in 61 independent hybrid clones. These results indicate that the gene coding for LDH-A is located on mouse chromosome 7.     

A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer

Summary A method is described to generate microcells from human lymphobalsts for use in microcell-mediated chromosome transfer (MMCT). Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated from these lymphoblasts by: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b) centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine kinase negative mouse fibroblast (LMTK⁻). HAT medium (hypoxanthine, aminopterin and thymidine) was used to select microcell hybrids expressing thymidine kinase activity. Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods this procedure permits microcells to be easily generated from lymphoid cells. This methodology of enucleation of microcells may be extended to a variety of other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones containing one or a few human chromosomes.     

A millipore--blue dextran affinity binding system.

Blue dextran has been adsorbed to millipore filter discs in a linkage stable under a variety of conditions. These discs have been to bind lactic dehydrogenase, which may then be specifically eluted with NADH, one of its substrates. In contrast, another enzyme, alkaline phosphatase, not expected to bind to blue dextran, was indeed completely recovered in the filtrate.     

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B 10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1⁺2^– and five Ly-1^–2⁺ T-cell clones were tested. The clones selected for the Ly-1 ⁺2^– phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-A^d or I-Ed). All Ly-1^–2^– clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (D^d and L^d). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and. function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - TCGF T cell growth factor (Interleukin-2) - Con A Concanavalin A - DME Dulbecco's modified Eagle's medium - PHA phytohaemagglutinin - LPS lipopolysaccharide - TRF-C T cell replacing factor required for induction of cytolytic cells from thymocytes - PBS phosphate-buffered saline (pH 7.4)     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

Linkage of the loci for glucose-6-phosphate dehydrogenase and for inosinic acid pyrophosphorylase to the X chromosome of the field-vole Microtus agrestis.

It has been proposed that there are strong selective pressures which have acted during the evolution of mammals to conserve the linkage of genes on the X chromosome. If so, loci that are known to be X-linked in one mammalian species should be X-linked in others. The loci for glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and for inosinic acid pyrophosphorylase (E.C. 2.4.2.8) are known to be X-linked in a variety of mammals. The linkage of these loci to the X chromosome of the field-vole, Microtus agrestis, is indicated by the pattern of segregation of these loci in hybrid cells derived by fusion of mouse cells with vole lymphocytes.     

Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.     

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142 g/L with production yield of 0.89 g/g and productivity of 3.55 g L⁻¹ h⁻¹ under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.     

cDNA Libraries from Single Human Preimplantation Embryos

In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10⁵and 10⁶clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, β-actin, keratin-18, and α-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 andAlurepeat sequences, housekeeping genes, and tissue-specific genes, such as α-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing “tissue-specific” genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Evidence for the inactivation of an X chromosome early in the development of the human female.

Studies of somatic tissues and cultured cells, including fibroblast clones, from human embryos heterozygous for the electrophoretic variants of glucose-6-phosphate dehydrogenase confirm that one X chromosome is inactivated very early in embryonic development and indicate that X inactivation has occurred in the majority of cells from a variety of tissues at least by 5 weeks from conception.