Salivary proline-rich protein genes on chromosome 8 of mouse

Endonuclease restriction (Hind III) fragments of DNA from Chinese hamster X mouse somatic cell hybrids hybridized with proline-rich protein complementary DNA clones only when the DNA was isolated from cells containing mouse chromosome 8, or a fragment of chromosome 8. The evidence suggests that proline-rich protein genes are located at the proximal portion of chromosome 8 toward the centromere.     

Induction of cellular senescence in immortalized cells by human chromosome 1

The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.     

Altering the genome by homologous recombination

Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.     

Interferon production and action in mouse, hamster and somatic hybrid mouse-hamster cells

A hybrid mouse-hamster cell line was developed from a mouse cell line which produces a high titer of interferon and is sensitive to its action, and a hamster cell line which produces little interferon and is relatively insensitive to its action. Parental cell lines demonstrated complete species specificity with respect to interferon production and action. The hybrid cells produced interferon (or interferons) effective when tested on the mouse cell line and primary hamster cells; the hybrids were sensitive to the action of both mouse and hamster interferons. Hybrid cells produced ten times more hamster interferon than the parent hamster cell line and were eight times more sensitive to hamster interferon than the parent hamster cell line.     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

Assignment of the gene for myelin proteolipid protein to the X chromosome: implications for X-linked myelin disorders

Several inherited disorders in humans and in rodents result in myelin dysgenesis and a deficiency of the molecular constituents of myelin. A complementary DNA to one of the two major myelin proteins, myelin proteolipid protein (also known as lipophilin), has been used with Southern blot analysis of somatic cell hybrid DNA to map the human proteolipid protein gene to the middle of the long arm of the human X chromosome (bands Xq13-Xq22) and to assign the murine proteolipid protein gene to the mouse X chromosome. Comparison of the gene maps of the human and mouse X chromosomes suggests that myelin proteolipid protein may be involved in X-linked mutations at the mouse jimpy locus and has implications for Pelizaeus-Merzbacher disease, a human inherited X-linked myelin disorder.     

Somatic cell hybrid between the established human line D98 (presumptive HeLa) and 3T3

Somatic cell hybrids have been made between an established human cell line with a long culture history and established mouse fibroblast line. When first analyzed, the hybrid cells contained nearly twice as many mouse chromosomes as the mouse parent line and a human chromosome complemnent of about half that of the human parent. There was further loss of human chromosomes on continued cultivation. This behavior resembles that of other human mouse hybrids and appears to be characteristic of the human-mouse combination. However, the number of human chromosomes is greater than in hybrids made from human diploid fibroblasts. Some clones contain more than a haptoid quantity of human DNA per cell and should synthesize a much greater number of human gene products.     

Primary structure and biochemical properties of an M2 muscarinic receptor

A partial amino acid sequence obtained for porcine atrial muscarinic acetylcholine receptor was used to isolate complementary DNA clones containing the complete receptor coding region. The deduced 466-amino acid polypeptide exhibits extensive structural and sequence homology with other receptors coupled to guanine nucleotide binding (G) proteins (for example, the beta-adrenergic receptor and rhodopsins); this similarity predicts a structure of seven membrane-spanning regions distinguished by the disposition of a large cytoplasmic domain. Stable transfection of the Chinese hamster ovary cell line with the atrial receptor complementary DNA leads to the binding of muscarinic antagonists in these cells with affinities characteristic of the M2 receptor subtype. The atrial muscarinic receptor is encoded by a unique gene consisting of a single coding exon and multiple, alternatively spliced 5' noncoding regions. The atrial receptor is distinct from the cerebral muscarinic receptor gene product, sharing only 38% overall amino acid homology and possessing a completely nonhomologous large cytoplasmic domain, suggesting a role for the latter region in differential effector coupling.     

Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts

Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.     

A new DNA marker tightly linked to the fragile X locus (FRAXA)

The fragile X syndrome is the most common cause of familial mental retardation. Genetic counseling and gene isolation are hampered by a lack of DNA markers close to the disease locus. Two somatic cell hybrids that each contain a human X chromosome with a breakpoint close to the fragile X locus have been characterized. A new DNA marker (DXS296) lies between the chromosome breakpoints and is the closest marker to the fragile X locus yet reported. The Hunter syndrome gene, which causes iduronate sulfatase deficiency, is located at the X chromosome breakpoint that is distal to this new marker, thus localizing the Hunter gene distal to the fragile X locus.     

Gene for T-cell growth factor: location on human chromosome 4q and feline chromosome B1

T-cell growth factor (TCGF) or interleukin-2 (IL-2), an immunoregulatory lymphokine, is produced by lectin- or antigen-activated mature T lymphocytes and in a constitutive manner by certain T-cell lymphoma cell lines. By means of a molecular clone of human TCGF and DNA extracted from a panel of somatic cell hybrids (rodent cells X normal human lymphocytes), the TCGF structural gene was identified on human chromosome 4. In situ hybridization of the TCGF clone to human chromosomes resulted in significant labeling of the midportion of the long arm of chromosome 4, indicating that the TCGF gene was located at band q26-28. Genomic DNA from a panel of hybrids prepared with HUT-102 B2 cells was examined with the same molecular clone. In this clone of cells, which produces human T-cell leukemia virus, the TCGF gene was also located on chromosome 4 and was apparently not rearranged. The homologous TCGF locus in the domestic cat was assigned to chromosome B1 by using a somatic cell hybrid panel that segregates cat chromosomes. Linkage studies as well as high-resolution G-trypsin banding indicate that this feline chromosome is partially homologous to human chromosome 4.     

普通小麦─大赖草6N二体异附加系的选育与鉴定

根据花粉母细胞减数分裂中期Ⅰ染色体配对情况及染色体Ｃ－分带，在中国春－大赖草杂种回交后代中选育出１个稳定的二体异附加系９２Ｇ４６０。通过分析亲本及９２Ｇ４６０的苗期叶片ＧＯＴ同工酶酶谱表型，推测９２Ｇ４６０中临时编为第１１号的大赖草染色体来自Ｎ染色体组，该染色体携有属于第６部分同源群的同工酶结构基因Ｇｏｔ－Ｎ２，故可以将大赖草中临时编为第１１号的染色体命名为６Ｎ。     

Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression

The development of Wilms' tumor, a pediatric nephroblastoma, has been associated with a deletion in the p13 region of chromosome 11. The structure and function or functions of this deleted genetic material are unknown. The role of this deletion in the process of malignant transformation was investigated by introducing a normal human chromosome 11 into a Wilms' tumor cell line by means of the microcell transfer technique. These variant cells, derived by microcell hybridization, expressed similar transformed traits in culture as the parental cell line. Furthermore, expression of several proto-oncogenes by the parental cells was unaffected by the introduction of this chromosome. However, the ability of these cells to form tumors in nude mice was completely suppressed. Transfer of other chromosomes, namely X and 13, had no effect on the tumorigenicity of the Wilms' tumor cells. These studies provide support for the existence of genetic information on chromosome 11 which can control the malignant expression of Wilms' tumor cells.     

转K2.9基因绒山羊体细胞核移植技术体系的优化研究

【目的】利用体细胞核移植技术制备转毛角蛋白Ⅱ型中间丝K2.9基因的高绒质绒山羊胚胎,为绒山羊优良品种的培育提供一种全新的技术材料。【方法】以含有Neor 基因标记的K2.9 毛囊特异表达载体pcDNA3.1-K转染绒山羊胎儿成纤维细胞,经G418筛选获得转K2.9基因细胞,将获得的转基因阳性细胞与体外成熟的绒山羊卵母细胞进行核移植,并对生产的重构胚进行了体外培养。本文分别进行了激活方法、供体细胞和卵母细胞来源的筛选,且对获得的囊胚进行了PCR鉴定。【结果】（1）Iono+6-D对成年羊卵母细胞的孤雌激活效果好于A23187+6-D,显著提高了胚胎的卵裂率。（2）羔羊孤雌胚的卵裂率显著低于成年羊,但囊胚率差异不显著。（3）来自2只绒山羊胎儿的转基因成纤维细胞对核移植胚的发育没有显著影响,但2号羊的转基因细胞显著提高了融合率。（4）以羔羊卵进行核移植,显著降低了核移植胚的发育率。（5）对获得的囊胚进行PCR鉴定,成功扩增到目的基因。【结论】将K2.9 毛囊特异表达载体pcDNA3.1-K转染的绒山羊胎儿成纤维细胞作为供体细胞,核移植到成年羊卵母细胞中,以Iono+6-D进行激活,首次成功、高效地获得携带K2.9基因的绒山羊囊胚。     

Chromosome mapping and expression of a putative testis-determining gene in mouse

Isolation and mapping of a mouse complementary DNA sequence (mouse Y-finger) encoding a multiple, potential zinc-binding, finger protein homologous to the candidate human testis-determining factor gene is reported. Four similar sequences were identified in Hind III-digested mouse genomic DNA. Two (7.2 and 2.0 kb) were mapped to the Y chromosome. Only the 2.0-kb fragment, however, was correlated with testis determination. Polymerase chain reaction analysis suggests both Y loci are transcribed in adult testes. A 3.6-kb fragment was mapped to the X chromosome between the T16H and T6R1 translocation breakpoints, and a fourth (6.0 kb) was mapped to chromosome 10. Hence, mYfin sequences have been duplicated several times in the mouse, although they are not duplicated in humans.     

Temporal coordination of DNA replication with enzyme synthesis in diploid and heteroploid cells

The rate of DNA synthesis in the S phase of growth of synchronized diploid Chinese hamster cells shows two maximums, while in heteroploid hamster cells the DNA replication rate is constant. In diploid cells a reciprocal relationship exists between maximum DNA synthetic rates and maximum lactate dyhydrogenase and thymidine kinase enzyme levels. Enzyme activity in heteroploid cells increases continuously through the cell cycle with no evidence of oscillations. It seems possible that these differences in molecular organization may accompany or precede the transition to heteroploidy.     

Animal cells: noncorrelation of length of G1 phase with size after mitosis

The interval between mitosis and initiation of DNA synthesis (G(1)) varied over a fourfold range for Chinese hamster ovary cells, an established line. This was not because of size differences. Synchlronous cells of different sizes began DNA synthesis at similar times after mitosis. A novel technique of centrifugation for separating cells according to size is described.