Characterization of the central region containing the X-inactivation center and terminal region of the mouse X Chromosome using irradiation and fusion gene transfer hybrids

The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.     

The human genetic map

The introduction of new technology and increased effort from around the world is driving the completion of the human gene map. In parallel with the creation of the map, we are beginning to see the bio-medical benefits that are a direct consequence of learning more about our own genome.     

Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin.

Activation of both T and B lymphocytes through their membrane receptors for antigen is known to induce breakdown of inositol phospholipids. In addition, T cell activation by antigen is accompanied by increased protein tyrosine phosphorylation of components of the T cell antigen receptor. We now provide evidence that B cell activation through membrane immunoglobulin is also coupled to stimulation of protein tyrosine kinase activity. One potential candidate for a B lymphocyte protein tyrosine kinase is an 80 kd molecule that is itself phosphorylated at tyrosine residues in response to stimulation with anti-immunoglobulin antibodies.     

Secretion of recombinant proteins from hydroxyethyl methacrylate-methyl methacrylate capsules

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate nonautologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immunoisolation devices before implantation would allow the use of the same recombinant cell line for treating different patients, thus potentially lowering the cost of treatment. To study the properties of a mechanically stable synthetic biomaterial, hydroxyethyl methyacrylate-methyl methacrylate (HEMA-MMA) as the immuno-isolation device, we encapsulated recombinant mouse fibroblast cells engineered to secrete products ranging from 27 to 300 kDa in size (human growth hormone, mouse beta-hexosaminidase and beta-glucuronidase) in the presence or absence of the extracellular matrix Matrigel. Both viability and cell number in the microcapsules increased with time after encapsulation and cell morphology indicated viable cell growth, thus showing that the capsule membrane barrier was compatible with nutrient/waste exchange necessary for normal metabolic activity.The intracellular levels of these recombinant gene products were constant throughout the experimental period of 22 days in the presence or absence of Matrigel, thus demonstrating that the microenvironment did not lead to downregulation of the transgenes. However, the extracellular levels of the gene products secreted from the cells and trapped within the microcapsules were dependent on the molecular size of the product and presence of Matrigel. With the 27-kDa human growth hormone, the presence of Matrigel caused its retention within this intracapsular space, but its release from the microcapsules to the culture medium was not impeded. With the 120-kDa beta-hexosaminidase or the 300-kDa beta-glucuronidase, they were retained within the microcapsule space regardless of the presence or absence of Matrigel, and their passage from the microcapsules to the media was totally blocked. In conclusion, the HEMA-MMA microcapsules are supportive of recombinant cell growth and maintained their molecular cutoff at approximately 100 kDa. Inclusion of extracellular matrix was unable to improve cell growth and may impede the exit of some gene products. (c) 1996 John Wiley & Sons, Inc.     

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.     

Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein.

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.     

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Analysis of a spatially regulated phosphotyrosine phosphatase identifies tyrosine phosphorylation as a key regulatory pathway in Dictyostelium.

We have cloned a Dictyostelium phosphotyrosine phosphatase (PTP1) with a catalytic domain showing approximately 38%-50% amino acid identity to those of other PTPs. PTP1 contains an approximately 99 amino acid insert and bacterially produced PTP1 possesses PTP activity. PTP1 is expressed at a very low level in vegetative cells, induced by 4 hr, and maximally expressed at the tight aggregate stage. PTP1-lacZ studies indicate that PTP1 is spatially localized to prestalk and anterior-like cell types. PTP1 gene disruptants show accelerated development, whereas strains overexpressing PTP1 to a high level fail to aggregate. Strains overexpressing moderate levels exhibit severe morphological defects following aggregation, including multiply tipped aggregates and morphologically aberrant fruiting bodies. Western blot analysis using anti-phosphotyrosine antibodies shows specific changes in the mutant strains when compared with wild-type cells. The results indicate that reversible protein-tyrosine phosphorylation and PTP1 play important regulatory roles during Dictyostelium development.     

Structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus.

We have found six major polypeptides in virions of the avian coronavirus infectious bronchitis virus grown in tissue culture: four glycoproteins, GP84, GP36, GP31, and GP28, and two non-glycosylated proteins, P51 and P23. In addition, we detected three minor species: two glycoproteins, GP90 and GP59, and one non-glycosylated protein, P14. Two-dimensional tryptic peptide mapping showed that GP36, GP31, GP28, and P23 comprise a group of closely related proteins which we have designated the "P23 family," but that the other proteins are distinct. Analysis by partial proteolytic digestion of P23 family, but that the other proteins are distinct. Analysis by partial proteolytic digestion of the P23 family labeled biosynthetically with [35S] methionine, and P23, labeled with [35S] formyl-methionine by in vitro translation of RNA from infected cells, revealed that the proteins of the P23 family differ in their amino-terminal domains. Similar analysis of GP31 and Gp36 labeled with [3H] mannose showed that the partial proteolytic fragments unique to these proteins were glycosylated. This suggests that differences in glycosylation in the amino-terminal domains contributes to the marked polymorphism os the P23 family. The results are discussed with respect to possible models for synthesis of the virion proteins.