Assignment of peptidase S (PEPS) to chromosome 4 in man using somatic cell hybrids

Summary A starch gel electrophoretic procedure is described that resolves peptidase S (PEPS) as well as the peptidases A, B, and C in man-rodent, rodent-rodent, and primate-rodent interspecific somatic cell hybrids. The interspecific PEPS cell hybrid phenotype can be resolved into a pattern which suggests that PEPS is composed of five or six identical subunits.Results are presented supporting assignment of the PEPS locus to chromosome 4 in man using man-mouse and man-Chinese hamster somatic cell hybrids. Human genes coding for peptidases A, B, C, and D were assigned to chromosome 18, 12, 1, and 19, respectively, confirming previous assignments. These somatic cell genetic data demonstrate the independent genetic control of the several human peptidases.This work was supported by NIH grants GM 20454 and HD 05196.     

The frequency and distribution of sister chromatid exchanges in human chromosomes

Summary A detailed procedure is described for a rapid detection of phosphoglucomutase-2 (=phosphopentomutase; PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells.The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.     

Starch gel electrophoretic phenotypes of mouse×human somatic cell hybrids and mouse isozyme polymorphisms

Summary Segregation of human chromosomes in man-mouse somatic cell hybrids affords a system for the linkage analysis of human gene loci. The isozymes constitute useful phenotypic markers for such studies, since homologous enzymes between man and mouse usually differ in their electrophoretic mobility. Electrophoretic techniques have been compiled for 22 isozymes. In this report, phenotypes are shown for mouse mouse-human somatic cell hybrids, and human cells cultured in vitro. Polymorphisms and linkage relationships are also discussed for inbred strains ofMus musculus. Supported by United States Public Health Service Grant GM-09966 from the Division of General Medical Sciences. Presented in the Symposium on Regulation in Tumor Cells at the Twenty-second Annual Meeting of the Tissue Culture Association.     

Direct assignment of citrate synthase (CS) gene to human chromosome 12 in man-mouse somatic cell hybrids

Summary A panel of twenty independently derived clones of man-mouse somatic cell hybrids isolated from fusions involving eight different parent cell combinations simultaneously analyzed for human chromosomes, citrate synthase, and a large number of other enzyme markers firmly or tentatively assigned to individual human chromosomes have provided direct evidence for a firm assignment of the structural gene coding for citrate synthase (CS) to human chromosome 12.     

Synthesis of ribosomal RNA in synkaryons and heterokaryons formed between human and rodent cells.

A study has been made of the ribosomal RNA and chromosome constitution of man-mouse hybrid cells. Previous work has shown that no human 28s rRNA is detectable in man-mouse synkaryons. In general human chromosomes are lost from such hybrids. With a recently developed method for distinguishing mouse from human chromosomes, an analysis of various man-mouse hybrid cell lines has been made. This indicates that not all the human chromosomes bearing nucleolar organizers are lost in the hybrid cells and such loss cannot alone explain the absence of human 28s rRNA. An examination of the 28s rRNA synthesized by heterokaryons formed from several different parent cells has revealed that both parental types of 28s rRNA are present in heterokaryons. The control of rRNA synthesis in hybrid cells is discussed.     

Control of lysosomal acid phosphatase expression in man-mouse cell hybrids

Lysosomal acid phosphatase activity in human and mouse cells was separated into multiple zones by starch gel electrophoresis. One of the two major zones in the mouse was apparently extinguished when genetic information from man and the mouse was combined in proliferating man-mouse somatic cell hybrids. The evidence suggested that the absence of the mouse lysosomal acid phosphatase (mAP-1) was influenced by the human genome. The gene coding for human acid phosphatase (hAP-1) was shown to be unlinked to the presumed human component which extinguished the mouse acid phosphatase (mAP-1). The mechanism of “extinction” is postulated to be a modification in the processing of the mouse lysosomal enzyme. A dimeric structure was suggested for acid phosphatase-1 of man, mouse, and rat since a single hybrid enzyme was expressed in man-mouse and mouse-rat somatic cell hybrids.     

Assignment of adenosine deaminase complexing protein (ADCP) gene(s) to human chromosome 2 in rodent-human somatic cell hybrids

Summary The experiments reported in this paper indicate that the expression of human adenosine deaminase complexing protein (ADCP) in the human-rodent somatic cell hybrids is influenced by the state of confluency of the cells and the background rodent genome. Thus, the complement of the L-cell derived A9 or B82 mouse parent apparently prevents the expression of human ADCP in the interspecific somatic cell hybrids. In the a3, E36, or RAG hybrids the human ADCP expression was not prevented by the rodent genome and was found to be proportional to the degree of confluency of the cell in the culture as in the case of primary human fibroblasts.An analysis of human chromosomes, chromosome specific enzyme markers, and ADCP in a panel of rodent-human somatic cell hybrids optimally maintained and harvested at full confluency has shown that the expression of human ADCP in the mouse (RAG)-human as well as in the hamster (E36 or a3)-human hybrids is determined by a gene(s) in human chromosome 2 and that neither chromosome 6 nor any other of the chromosomes of man carry any gene(s) involved in the formation of human ADCP at least in the Chinese hamster-human hybrids. A series of rodent-human hybrid clones exhibiting a mitotic separation of IDH1 and MDH1 indicated that ADCP is most probably situated between corresponding loci in human chromosome 2.A part of the results was presented at the Fifth International Conference on Human Gene Mapping, Edinburgh, July 1979 and reported as an abstract in the proceedings of this conference [Cytogenet Cell Genet 25:164 (1979)]     

Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids

We have analysed metaphase chromosomes of man-mouse somatic cell hybrids using a sequential staining procedure involving the fluorescent DNA-binding stains, Hoechst 33258 and quinacrine mustard. This was found to be a simple and reliable means of differentiating the chromosomes of the two species and of identifying specific human chromosomes. In addition, this method will permit the study of the segregation of human chromosome homologues that are discordant for quinacrine mustard fluorescent polymorphisms.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

IL9 maps to mouse chromosome 13 and human chromosome 5

Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL-9 is distal to Tcrg and Proximal to Dhfr.     

Mapping of the gene for anti-müllerian hormone to the short arm of human chromosome 19

The gene coding for human anti-Müllerian hormone (AMH) was localized to subbands p13.2----p13.3 on chromosome 19, using in situ hybridization and Southern blot analysis of a panel of man-mouse and man-hamster somatic cell hybrids.     

Human mitochondrial NADP-dependent isocitrate dehydrogenase in man-mouse somatic cell hybrids.

Human mitochondrial NADP-dependent isocitrate dehydrogenase (IDH-2) is expressed in man-mouse somatic cell hybrids as a dimeric molecule. The gene specifing this enzyme was observed to be syntenic with the mannose phosphate isomerase locus in the 56 primary man-mouse clones in this series. The human IDH-2 locus, therefore, may be assigned to chromosome 15.     

Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain.

Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids.     

Quantitative analysis of human chromosome segregation in man-mouse somatic cell hybrids.

The presumed random and independent process of human chromosome segregation in man-mouse somatic cell hybrids was studied. The results of chromosome analysis on 196 cells from 15 related hybrid strains have provided the first convincing evidence that segregation of human chromosomes can be nonindependent and often concordant. Different human chromosomes were not retained with equal frequency in these hybrid clones. Some were present in 80% of all the cells, whereas others appeared in less than 10% of the same cells. Linear regression analysis was used to test for correlation of the frequencies of all pair-wise combinations of human chromosomes present in these hybrid clones. Twenty-two of 136 possible correlations were statistically significant, indicating that concordant segregation of particular pairs of human chromosomes is a rather frequent occurrence.     

Mapping AK1, ACONs, and AK3 to chromosome 9 in man employing and X/9 translocation and somatic cell hybrids.

The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.     

Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter)

The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter.     

Assignment of a fibronection gene to human chromosome 2 using monoclonal antibodies

The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin, Both monoclonal antibodies demonstrate a highly concordant association between the presence of two different human fibronectin antigens and human chromosome 2.     

A glutaminase (gis) gene maps to mouse chromosome 1, rat chromosome 9, and human chromosome 2

A rat cDNA clone encoding a portion of phosphate-activated glutaminase was used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between wild-derived and inbred strains of mice. Segregation of rat and mouse chromosomes among somatic cell hybrids indicated assignment to rat chromosome 9 and mouse chromosome 1. Analysis of chromosome 1 alleles for several genes in an interspecific cross between Mus spretus and C3H/HeJ-gld/gld mice indicates that glutaminase can be positioned within 5.5 +/- 2.0 cM proximal to Ctla-4. Similarly, human-hamster somatic cell hybrids were examined for RFLPs, and four human EcoRI restriction fragments were found to hybridize with the rat glutaminase probe. Two of these restriction fragments cosegregated and mapped to human chromosome 2 in a region that is syntenic with mouse chromosome 1 and rat chromosome 9.     

Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen

Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene.     

Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes

Constitutive heterochromatin of chromosomes can be visualized utilizing a new differential staining technique which was originally developed by Gall and Pardue (1971). The method facilitates the more certain identification of specific chromosomes within and between cell populations of different origins. Marker chromosomes can be identified in established cell lines over many months of serial passage. Chromosomes of similar morphology within karyotypes of man and mouse can be distinguished in a number of instances. For example, the Y chromosomes of both mouse and man can now be easily detected. The hetero-chromatic staining method also permits discrimination between mouse and human chromosomes in somatic cell hybrids, thus facilitating the assignment of gene markers to chromosomes in somatic cell genetics systems. Instances of translocation of centric heterochromatin to other parts of chromosomes in established tissue culture cell lines are described. An instance of the inheritance of a polymorphic variation in autosomal heterochromatin in man is reported. It is postulated that polymorphisms in the centric heterochromatin may account largely for small heritable chromosome length variations previously described in human populations and termed minor chromosome variants.