Somatic Cell Genetics Research Articles

A genetic analysis of the adenine phosphoribosyl transferase locus in Chinese hamster V79-AP4 cells: relevance to mutagenesis studies

The chromosomal location of the autosomal locus aprt has been investigated in the permanent Chinese hamster cell line V79-AP4 by standard somatic cell genetics methodologies. Aprt is functionally dizygous in V79-AP4 and the a alleles map on 2 chromosome 3 homologs, in agreement with the chromosome assignment of the gene in Chinese hamster primary cells. Chromosome G-banding and a Southern blot analysis of V79-AP4 DNA, using as a probe the cloned Chinese hamster aprt gene, have not revealed any structural alteration at either of the 2 aprt alleles. One of the chromosomes 3 has, however, a terminal deletion in its long arm and is therefore morphologically marked. These findings could make V79-AP4 an interesting cell system for the study of mutational mechanisms at the aprt locus in Chinese hamster.

Plant Regeneration from Strawberry ( Fragaria x Ananassa) Mesophyll Protoplasts

In vegetatively propagated species with strong heterozygoty and high ploidy levels, plant breeding through somatic cell genetics is of special interest. In strawberry breeding, where plant breeders wish to introduce specific traits such as earliness, winterhardiness and disease resistance into already established cultivars these techniques might be of great value. A prerequisite for somatic hybridization and direct gene transfer is an efficient procedure for isolation of protoplasts and plant regeneration. In this paper we present a method for protoplast isolation and plant regeneration in strawberry, a species which we intend to use in a coming fusion project.

Cell Culture and Somatic Cell Genetics of Plants. Volume 4: Cell Culture in Phytochemistry.Friedrich Constabel , Indra K. Vasil

<i>Cell Culture and Somatic Cell Genetics of Plants. Volume 4: Cell Culture in Phytochemistry.</i>Friedrich Constabel , Indra K. Vasil

Phenotypic cyclophosphamide resistance in mouse myeloma hybridomas.

We have analyzed the expression of cyclophosphamide (CY) resistance in somatic cell hybrids of mouse LPC-1/CY-R plasmacytoma and P3-X63-Ag8.653 (Ag8) myeloma cells. LPC-1/CY-R tumor is resistant to curative doses (60-250 mg/kg body weight) of CY. The median survival time (MST) of drug treated LPC-1/CY-R tumor bearing mice is 25 days, similar to that of untreated mice. LPC-1/CY-R tumor cells secrete an IgG 2a kappa M component and do not survive in tissue culture. Ag8 tumor cells are CY sensitive, are selected out in hypoxanthine-aminopterin-thymidine (HAT) media and do not secrete any immunoglobulin. Hybrids formed between these two cell lines survived in HAT and secreted IgG 2a kappa. Hybrid cells exhibited greater ploidy than that of their parents and contained the metacentric marker chromosome of the Ag8 cell line. Hybrid cells exhibited the same growth characteristics in BALB/c mice as that of their LPC-1/CY-R parent and were resistant to curative doses of CY. These studies demonstrate that, CY resistance is a somatic cell dominant trait which can be transferred to daughter cells via somatic cell hybridization. Availability of somatic cell hybrids produced between CY sensitive and resistant tumor cells may provide useful tools to study the biochemical nature and the somatic cell genetics of this drug resistant trait.

Development and characterization of mutant chicken cell lines for somatic cell genetics studies.

A series of stable mutants bearing nuclear genetic markers were developed from the established chicken cell line DU24. The mutants were obtained after mutagenesis of DU24 cells with ethyl methanesulfonate (EMS) or arose spontaneously when plated in the appropriate selective medium. Clones resistant to 5-bromodeoxyuridine (BrdU) were obtained following a two-step selection procedure and analyzed. The BrdUr cells were found to be deficient in thymidine kinase activity and were HAT sensitive. Molecular characterization of these mutants revealed no deletions or other rearrangements, but methylation of some cytosine residues was decreased in the mutants. A similar restriction profile was seen in a series of mutants made resistant to BrdU after cultivation of DU24 cells in increasing concentrations of the drug over a period of six months. Selection of EMS-treated BrdUr cells in 10 microM ouabain gave rise to a clone resistant to both drugs and which was still HAT sensitive. Clones resistant to 6-thioguanine were also isolated, but showed wild-type hypoxanthine phosphoribosyltransferase activity and were HAT resistant. A number of the cell lines isolated were found to be suitable for fusion experiments with both chicken cells and cells from other vertebrate species.

Molecular Cytogenetics of Cattle: A Genomic Approach to Disease Resistance and Productivity

Classical cytogenetics has been merged with somatic cell and molecular genetics to facilitate mapping of the bovine genome. A physical map is presently being generated by the use of hybrid somatic cells and in situ hybridization to assign genes to chromosomes. A complementary genetic map is being generated by analysis of recombination of polymorphic loci, many of them identified with the same cloned DNA probes used for physical mapping. The eventual utilization of this map for selective breeding of disease resistance and productivity is dependent on the saturation of the map with polymorphic markers at a density that will assure linkage of genes influencing desirable phenotypes with at least one polymorphic marker. The identification of regions of chromosomal conservation among cattle, mice, and humans is being exploited to facilitate the saturation of the bovine map with useful markers.

Mapping of bovine PRGS and PAIS genes in hybrid somatic cells: Syntenic conservation with human chromosome 21

Somatic cell genetics coupled with enzyme electrophoresis has facilitated the mapping of PRGS and PAIS genes in cattle. Individual cow-hamster hybrid cell lines established by fusion of mutant CHO cells, ade-C and ade-G, with cattle leukocytes required complementing bovine genes for PRGS and PAIS, respectively, when propogated on selective media. Homogenates of 12 PRGS+ hybrid clones and 12 PAIS+ hybrid clones retained the bovine electromorph of SOD1 while extensively segregating 14 biochemical markers of other cattle syntenic groups. Secondary cattle-hamster hybrid subclones which segregated bovine PRGS and PAIS in late passages on nonselective media concordantly segregated bovine SOD1. These data support a syntenic relationship among PRGS, PAIS, and SOD1 on cattle syntenic group U10. An interferon receptor locus, IFREC, is also syntenic with SOD1. This synteny represents an extensive conservation of bovine U10 and the "Down syndrome region" of human chromosome 21.

Gene regulation, organization and function

A recent meeting ★ ★ The FASEB Conference on Somatic Cell Genetics was held at Saxtons River, Vermont, USA, 3–8 July 1987. on somatic cell genetics concentrated heavily upon long range gene organization, the production of long range genetic maps and the use of mutagenesis to probe gene function in development. But the meeting also heard how the application of molecular approaches to gene regulation, organization and function has encroached far into immunological territory.

Selecting in Higher Plant Cell Cultures. A Discussion

Summary Procedures of somatic cell genetics provide additional methods for manipulation in plant breeding which must, however, be standardized to be used effectively and successfully. The expectations connected with the employment of selection in higher plant cell cultures have not been fulfilled yet. Undoubtedly, this is mainly due to the lack of adapted selection procedures. Cell cultures of higher plants possess qualities that have to be taken into account when elaborating selection procedures: (1) cell suspensions mostly contain cell colonies; (2) higher plant cell cultures are characterized by genetic instability; (3) cell cultures do not form expected traits, but exhibit traits in another form or develop new ones; (4) useful individual plants have to be regenerated from single cells in most cases; (5) plating efficiency is reduced; (6) spontaneous selection processes may diminish the range of traits selectable from cell cultures. These problems as well as advantages and drawbacks of selection procedures in current use are discussed.

Cell Culture and Somatic Cell Genetics of Plants. Volume 3: Plant Regeneration and Genetic Variability. Indra K. Vasil

Cell Culture and Somatic Cell Genetics of Plants. Volume 3: Plant Regeneration and Genetic Variability. Indra K. Vasil

Analysis of human chromosome 11 by somatic cell genetics: reexamination of derivatives of human-hamster cell line J1.

Through the fusion of a CHO cell population to a human cell population, a hybrid cell line which has lost all human chromosomes except chromosome 11 was derived. This cell line, J1, does not appear to segregate human chromosome 11 during growth. A series of deletion segregants were isolated from J1 which had lost a portion of either the long, short, or both arms of chromosome 11. This panel of deletion segregants was used for mapping a number of genetic markers on the short arm of chromosome 11. Karyotypic analysis led to the interpretation that derivatives of J1 selected for the loss of cell surface antigens encoded by genes on the short arm of the chromosome had simple terminal deletions of this chromosome arm. More recently, we have applied recombinant DNA and in situ hybridization techniques to the analysis of the structure of chromosome 11. In the course of this analysis, we have obtained data that indicate that all J1 deletion segregants retain a small chromosomal segment containing the structural genes for insulin and HRAS1. Analysis of in situ hybridization data indicates that in cell lines in which a chromosome 11 fragment cannot be identified by karyotype analysis, human DNA has been translocated to a Chinese Hamster chromosome. These results suggest that the original interpretation of the karyotypes of deletion segregants derived from J1 as simple terminal deletions is not correct. A reanalysis of gene localization studies based on these deletion segregants suggests that some assignments of genes to specific bands on chromosome 11 should be reconsidered. In particular, data on additional deletion segregants are consistent with localization of the beta-globin gene complex to band 11p15. The data presented here suggest that in several hybrid derivatives of J1, a continuous DNA segment of approximately 10(7) base pairs in length which includes the insulin and HRAS1 (cellular homolog of retroviral oncogene Harvey ras) genes has been isolated from the remainder of the human genome. We propose that the stability of chromosome 11 in the original hybrid was due to complementation of a genetic defect in the original CHO cell parent by a gene located in close physical proximity to the insulin and HRAS1 genes on chromosome 11. Data are presented which test and support this hypothesis.

Genetics in Medicine

Genetics of Man, by F. Clarke Fraser and James J. Nora, ed 2; 352 pp, with illus, $31, Philadelphia, Lea & Febiger, 1986. Thompson and Thompson's Genetics in Medicine has established itself as a useful text for medical students and physicians having no prior knowledge of genetics. The purpose is introduce medical students to the principles, language and methods of human genetics and to indicate some of its actual and potential medical significance. This edition, the first revised solely by Margaret W. Thompson, continues a successful tradition. The format remains that of previous editions. Initially, chapters are devoted to reviewing principles of medical genetics: cytogenetics and the clinical spectrum of chromosomal aberrations; single-gene inheritance, illustrated by hemoglobinopathies and metabolic disorders; molecular genetics; and multifactorial inheritance. A series of topics is then succinctly covered as separate chapters: immunogenetics, blood grouping, somatic-cell genetics, population genetics, twins, developmental genetics, linkage and mapping,

The role of chromosomal translocations in B- and T-cell neoplasia.

At the turn of the century, Boveri (1) suggested that abnormal chro­ mosomal patterns in tumor samples reflect the processes responsible for the development of the malignant phenotype. However, the first demonstrated association of a consistent chromosomal rearrangement with a specifi c disease did not occur until 1960 when a marker chromosome, the Phi­ ladelphia chromosome (Ph +), was discovered to be consistently associated with chronic myelogenous leukemia (CML) (2). The Ph+ chromosome was subsequently shown to result from a reciprocal translocation between chromosomes 9 and 22 (3). Due to the introduction of new techniques for high resolution ban­ ding-analysis of chromosomes, a large majority of human cancer cells are now known to carry clonal cytogenetic changes (4). This has been most clearly demonstrated for the hematopoietic malignancies and has resulted in a renewed focus on chromosomal rearrangements as a causative factor in malignant transformation. While this view was originally not widely accepted outside of the discipline of cytogenetics, the painstaking efforts to observe and catalogue these rearrangements have now been justifi ed. The application of the new technologies of molecular biology and somatic cell genetics have allowed the characterization of a variety of chromosomal abnormalities, and in particular chromosomal translocations, at the mol-

HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.

Genetic analysis of the human Y chromosome by chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to segregate fragments of human chromosomes in human-rodent hybrid cells. As with all somatic cell genetics methods, a selection technique is needed to isolate the hybrid cell lines produced by CMGT. Expression of the MIC2 gene product on the cell surface (the 12E7 antigen) provides an endogenous selectable marker for the human Y chromosome. Using chromosome transfer followed by separation of 12E7 antigen-positive cells on the fluorescence-activated cell sorter, a series of cell lines containing segregated fragments of the Y chromosome have been derived. The possibility of using these fragments to derive fine structural mapping data for the Y chromosome is considered in this review.

A new moderately repetitive DNA sequence family of novel organization.

In cloning adenovirus homologous sequences, from a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digests of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (4q31) and 19 (q13.1-q13.5). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.

Assignment of proteins to human chromosome 21 using two-dimensional gel electrophoresis and somatic cell genetics: An approach to the study of Down syndrome

A mouse hybrid cell line (WA17d) was derived which contained multiple copies of human chromosome 21 and no other human chromosome. Cell extracts of this line were prepared and subjected to two-dimensional gel electrophoresis. Several proteins were identified whose synthesis was altered by the presence of chromosome 21 and 6 proteins were identified as being specific to this human chromosome. These gene products might be involved in the pathogenesis of Down syndrome and be related to the neurologic defects and premature aging seen in this common chromosome abnormality.

Assignment of proteins to human chromosome 21 using two-dimensional gel electrophoresis and somatic cell genetics: An approach to the study of Down syndrome

A mouse hybrid cell line (WA17d) was derived which contained multiple copies of human chromosome 21 and no other human chromosome. Cell extracts of this line were prepared and subjected to two-dimensional gel electrophoresis. Several proteins were identified whose synthesis was altered by the presence of chromosome 21 and 6 proteins were identified as being specific to this human chromosome. These gene products might be involved in the pathogenesis of Down syndrome and be related to the neurologic defects and premature aging seen in this common chromosome abnormality.

Translocation of oncogene c-sis from chromosome 22 to chromosome 17 in a human myelogenous leukemia cell line.

By combining somatic cell genetics, in situ hybridization and Southern hybridization, we found that the c-sis oncogene in the human myelogenous leukemia cell line ML3 is translocated from the long arm (q11----qter) of chromosome 22 to the long arm (mid-portion or q21 region) of chromosome 17. This translocation does not result in rearrangement of the c-sis oncogene.

The Definitive Cookbook Cell Culture and Somatic Cell Genetics of Plants: Laboratory Procedures and Their Applications, Vol. 1 Indra K. Vasil

The Definitive Cookbook Cell Culture and Somatic Cell Genetics of Plants: Laboratory Procedures and Their Applications, Vol. 1 Indra K. Vasil