PubMed Health⌕ Search

Biomedical subjects

A Berns

Publications and source records attributed to A Berns.

At least 19 recordsLinked to original sources
PubMed1992

Requirement for a functional Rb-1 gene in murine development.

Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.

Abnormalities, Multiple↗
PubMed1992

T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation.

A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.

Animals↗
PubMed1992

Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.

A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergences between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.

Animals↗
PubMed1992

The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation.

We mutagenized, mapped, and sequenced the pseudorabies virus (PRV) homology of gene UL21 of herpes simplex virus type 1. A polyclonal mouse antiserum against the protein encoded by the UL21 homolog was generated and used to monitor the expression and subcellular localization of the UL21-encoded protein. We found that the protein is identical to a previously detected PRV capsid protein. We analyzed viable PRV strains encoding mutant UL21 homologys, truncated by insertion of an oligonucleotide that contains stop codons in all reading frames. In two PRV mutants carrying the oligonucleotide at two sites within the gene, processing of newly replicated viral DNA was impaired. In addition, we show that one of the UL21 mutants has strongly reduced virulence for mice.

Amino Acid Sequence↗
PubMed1992

Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells.

We have performed large-scale random oligonucleotide insertion mutagenesis on a 41-kbp genomic segment derived from the unique long (UL) region of the alphaherpesvirus pseudorabies virus (PRV). This procedure has resulted in the generation of a series of PRV strains, each carrying a single gene whose termination of translation is induced by the inserted oligonucleotide. To relate the genes that were involved in the mutagenization to genes previously identified in herpes simplex virus type 1, the prototype alphaherpesvirus, we have performed cross-hybridization studies. In this way, we have mapped the location of the homolog of a gene which was described to have sequence characteristics of a eukaryotic phosphotransferase. We characterized the phenotype of a mutant PRV strain lacking this putative phosphotransferase also the phenotype of a PRV strain lacking, in addition to the UL-encoded putative phosphotransferase, the protein kinase encoded within the unique short region of the virus. To assess the enzymatic activity of the UL region-encoded phosphotransferase, we expressed the gene transiently in a eukaryotic expression system. Immunoprecipitation of the protein followed by kinase assays and phosphoamino acid analyses revealed protein-serine/threonine kinase activity. Implications of sequence divergence of this protein from classical protein-serine/threonine kinases for kinase structure and function are discussed in view of the recent resolution of the structure of the catalytic domain of cyclic AMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1991

Sequence similarity between the mammalian bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2.

The bmi-1 proto-oncogene can be activated by Moloney murine leukaemia proviral insertions in E mu-myc transgenic mice. It encodes a highly conserved nuclear protein of 324 amino acids which belongs to a family of proteins containing a putative new zinc-finger. Another closely related member of this family is the mouse protein Mel-18. Here we report on the cloning and characterization of a homologous gene (D-bmi) from Drosophila melanogaster. Our analysis indicates that distinct domains of the mouse Bmi-1 protein, including the putative zinc-finger motif, are highly conserved within the much larger D-Bmi protein. Chromosomal localization and sequence comparison reveal that D-bmi is identical to Posterior Sex Combs (Psc) and indicate that the conserved domains between mouse bmi and Psc are also conserved within Suppressor-2 of Zeste (Su(z)2).

Amino Acid Sequence↗
PubMed1991

A pgk::hprt fusion as a selectable marker for targeting of genes in mouse embryonic stem cells: disruption of the T-cell receptor delta-chain-encoding gene.

We have constructed a hypoxanthine phosphoribosyl transferase-selectable marker (hprt) under the control of the phosphoglycerate kinase (pgk) promoter. This construct permits cell growth in hypoxanthine/aminopterin/thymidine media and confers 6-thioguanine sensitivity upon mouse Hprt- embryonic stem cells, allowing either positive or negative selection in gene-targeting experiments. We have successfully targeted the gene encoding the T-cell receptor delta-chain using the pgk::hprt fusion for counterselection.

Animals↗
PubMed1991

Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging.

Mo-MLV infection of E mu-myc transgenic mice results in a dramatic acceleration of pre-B cell lymphomagenesis. We have used provirus tagging to identify genes that cooperate with the E mu-myc transgene in B cell transformation. Here we report on the identification of four loci, pim-1, bmi-1, pal-1, and bla-1, which are occupied by proviruses in 35%, 35%, 28%, and 14% of the tumors, respectively. bmi-1, pal-1, and bla-1 represent novel common proviral insertion sites. The bmi-1 gene encodes a 324 amino acid protein with a predominantly nuclear localization. bmi-1 is highly conserved in evolution and contains several motifs frequently found in transcriptional regulators, including a new putative zinc finger motif. No genes have yet been assigned to pal-1 and bla-1. The distribution of proviruses over the four common insertion sites suggests that provirus tagging can be used not only to identify the cooperating oncogenes but also to assign these genes to distinct complementation groups in tumorigenesis.

Amino Acid Sequence↗
PubMed1991

Carcinogen-induced lymphomagenesis in pim-1 transgenic mice: dose dependence and involvement of myc and ras.

Transgenic mice overexpressing the pim-1 oncogene in their lymphoid compartments are predisposed to T-cell lymphomagenesis but only to the extent that approximately 10% of the transgenic mice develop lymphomas within 34 weeks after birth. Recently, we have shown that lymphomagenesis in pim-1 transgenic mice can be accelerated by infecting pim-1 transgenic mice with murine leukemia viruses or by treating the mice with a relatively low dose of 60 mg of the carcinogen N-ethyl-N-nitrosourea (ENU) per kg of body weight. Here we describe the incidence of tumors as a function of the dose of ENU. Either 200, 15, 4, 1, or 0.1 mg/kg ENU was injected into transgenic and control mice and the tumor incidence was monitored. T-cell lymphomas developed in 100 and 70% of the pim-1 transgenic mice treated with 200 and 15 mg/kg ENU, respectively. Approximately 20% of the Emu-pim-1 transgenic mice developed lymphomas after treatment with either 4, 1, or 0.1 mg/kg ENU. The nontransgenic mice developed lymphomas only after injection with 200 mg/kg (45%). The data show that Emu-pim-1 transgenic mice are approximately 25-fold more susceptible to ENU-induced lymphomagenesis than control mice. In most tumors the expression of c-myc was strongly elevated, probably as a direct or indirect effect of ENU. Analysis of the lymphomas for ras mutations revealed that approximately 10% of the lymphomas bear a ras mutation. The data indicate that at least some of these mutations are not the direct result of alkylation by ENU but rather represent spontaneous mutations that occurred later in the tumorigenic process.

Animals↗
PubMed1991

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.

Amino Acid Sequence↗
PubMed1991

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera.

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).

Animals↗
PubMed1991

Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally.

Previously, it has been shown that E mu-pim-1 transgenic mice are predisposed to T-cell lymphomas, whereas E mu-myc transgenic mice are predisposed to pre-B-cell lymphomas. Here we show that double-transgenic E mu-myc E mu-pim-1 mice exhibit pre-B-cell leukemia in utero. Upon transplantation into recipient mice, embryo-derived double-transgenic leukemic cells frequently progressed to highly malignant monoclonal tumors, indicating that additional (epi)genetic events had occurred during the progression of the disease.

Animals↗
PubMed1991

E mu N- and E mu L-myc cooperate with E mu pim-1 to generate lymphoid tumors at high frequency in double-transgenic mice.

Transgenic mice that contain the L-myc gene under the control of the immunoglobulin heavy-chain enhancer (E mu) express the transgene preferentially in T cells, develop thymic hyperplasia and are predisposed to T-cell lymphomas. An analogous E mu N-myc transgene is expressed preferentially in pre-B and B cells and provokes the development of B-cell neoplasias. Animals with an E mu pim-1 construct express the transgene in both B and T cells, but succumb to T-cell lymphomas. Complementation of the E mu N- and L-myc transgenic mice by breeding with E mu pim-1 animals leads to much more rapid development and a dramatically higher incidence of lymphoid malignancies, but the lineage specificity prescribed by the E mu N- and L-myc transgenes is maintained. The different oncogenic potential of myc genes is illustrated by the average latency period of tumor manifestation in double transgenics. Whereas c-myc/pim-1 animals develop pre-B-cell leukemia prenatally, the mean latency period for N-myc/pim-1 and L-myc/pim-1 mice is 36 and 94 days respectively. The N- and L-myc transgenes are expressed at high levels in tumors from double transgenic mice, but expression of the endogenous c- and N-myc genes is undetectable, directly implicating the myc transgenes in the tumor formation process.

Animals↗
PubMed1990

Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells.

Specific genes can be inactivated or mutated in the mouse germ line. The phenotypic consequences of the mutation can provide pivotal information on the function of the gene in development and maintenance of the mammalian organism. The procedure entails homologous recombination in embryonic stem cells, which, on fusion to recipient blastocysts, give rise to chimaeric mice that can transmit the mutant gene to their offspring. Inbreeding can then yield mice carrying the mutation in both alleles allowing the phenotypic analysis of recessive mutations. In addition to mice lacking a particular gene function, cell lines carrying null alleles of normally expressed genes can be instrumental in assessing the function of the gene. These cell lines can either be obtained from homozygous animals or, should the mutation be lethal early in embryonic development, be generated by consecutive inactivation of both alleles by homologous recombination in cultured cells. Here we illustrate the feasibility of this latter approach by the efficient consecutive inactivation of both alleles of the pim-1 proto-oncogene in embryonic stem cells.

Animals↗