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G Calothy

Publications and source records attributed to G Calothy.

At least 55 records · Page 3Linked to original sources
PubMed1993

Transcriptional downregulation of the retina-specific QR1 gene by pp60v-src and identification of a novel v-src-responsive unit.

The embryonic avian neuroretina (NR) is part of the central nervous system and is composed of various cell types: photoreceptors and neuronal and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of differentiating NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-src gene. To understand the mechanisms involved in the regulation of neural cell growth and differentiation, we studied the transcriptional regulation of QR1, a gene specifically expressed in postmitotic NR cells. Transcription of this gene is detected primarily in Müller cells and is strongly downregulated by the v-src gene product. Moreover, QR1 expression takes place only during the late phase of retinal development and is shut off abruptly at hatching. We have isolated a promoter region(s) of the QR1 gene that confers v-src responsiveness. By transfection of QR1-CAT constructs into quail NR cells infected with the temperature-sensitive mutant of RSV, PA101, we have identified a v-src-responsive region located between -1208 and -1161 upstream of the transcription initiation site. This sequence is able to form two DNA-protein complexes, C1 and C2. Formation of complex C2 is specifically induced in cells expressing an active v-src product, while formation of C1 is detected mainly in nonproliferating quail NR cells upon pp60v-src inactivation. C1 is also a target for regulation during development. We have identified the DNA binding site for the C1 complex, a repeated GCTGAC sequence, and shown that mutations in this element abolish binding of this factor as well as transcription of the gene at the nonpermissive temperature. Neither formation of C1 nor that of C2 seems to involve factors known to be targeted in the pp60v-src cascade. Our data suggest that C1 could be a novel target for both developmental control and oncogene-induced cell growth regulation.

Actins↗
PubMed1993

Serum factors and v-src control two complementary mitogenic pathways in quail neuroretinal cells in culture.

Quail neuroretinal cells (QNR cells) from 7-day-old embryos do not proliferate even in the presence of 8% fetal calf serum. After infection by the Rous sarcoma virus (RSV) they proliferate actively and exhibit a transformed phenotype; this effect is mediated by the oncoprotein pp60v-src. Secondary cultures infected by the thermosensitive strain tsNY68 of RSV are blocked in G0 either by thermal inactivation of pp60v-src at 41.5 degrees C or by serum deprivation at the permissive temperature (36.5 degrees C). Cell division is reinduced either by pp60v-src thermal renaturation or by subsequent serum addition. Our results indicate that v-src and serum control two synergic pathways leading to G0/G1 transition in QNR cells. In order to characterize genes related to the mitogenic and transforming effects of v-src in nerve cells, we have constructed a cDNA library from QNR cells transformed by tsNY68. We report the properties of five molecular clones isolated by differential screening of this library. Unlike immediate-early genes like c-fos, they are induced in mid and late G1. Four of them correspond to unknown mRNAs and the last one codes for nucleolin. This set of v-src-regulated genes is likely to code for functions deficient in terminally differentiated QNR cells and necessary for the progression in G1.

Animals↗
PubMed1992

Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties.

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.

Amino Acid Sequence↗
PubMed1992

Quail neuroretina c-Rmil(B-raf) proto-oncogene cDNAs encode two proteins of 93.5 and 95 kDa resulting from alternative splicing.

c-Rmil is the cellular allele of the v-Rmil oncogene transduced during in vitro passaging of Rous-associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmil proto-oncogene is the avian homolog of the mammalian B-raf gene and belongs to the mil/raf oncogene family of serine/threonine protein kinases. The c-Rmil/B-raf gene is preferentially expressed in avian and mammalian neural tissues. Two c-Rmil cDNA species, resulting from an alternative splicing mechanism, were isolated from quail embryonic NR cDNA libraries. They encode two proteins of 767 and 807 amino acids that differ by the presence of an alternative exon, located upstream of the kinase domain. Expression of these cDNAs in COS-1 cells leads to the synthesis of two proteins with apparent molecular weights of 93.5 and 95 kDa, recognized by an Rmil-specific antiserum. Both proteins are phosphorylated in an immune complex kinase assay. A protein of 94 kDa is also immunoprecipitated in avian NR cells and is identical to the 93.5-kDa protein expressed in COS-1 cells, as shown by Staphylococcus aureus V8 protease mapping. The c-Rmil proteins contain the three conserved regions previously identified in mil/raf protein kinases. In addition, they contain amino-terminal sequences that are not present in the other mil/raf proteins identified to date. These additional sequences may define a novel functional domain for c-Rmil/B-raf and could play a role in signal transduction in neural cells.

Amino Acid Sequence↗
PubMed1992

Chromosomal assignment of two human B-raf(Rmil) proto-oncogene loci: B-raf-1 encoding the p94Braf/Rmil and B-raf-2, a processed pseudogene.

The B-raf gene is the human homolog of the avian c-Rmil proto-oncogene encoding a 94-kDa serine/threonine kinase detected in avian cells. We have previously shown that this protein contains amino-terminal sequences not found in other proteins of the mil/raf gene family. These sequences are encoded by three exons in the avian genome. We report that these three exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the avian c-Rmil gene, indicating that in both avian and mammalian species the product of the B-raf/c-Rmil gene is a 94-kDa protein. We also identified two human B-raf loci: B-raf-1, located on chromosome 7q34, which encodes the functional B-raf/Rmil gene product, and B-raf-2, an inactive processed pseudogene located on chromosome Xq13.

Amino Acid Sequence↗
PubMed1991

Transcription of a quail gene expressed in embryonic retinal cells is shut off sharply at hatching.

The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.

Amino Acid Sequence↗
PubMed1991

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

Amino Acid Sequence↗
PubMed1991

Cell division is required for expression of v-myc transforming properties in chicken embryonic neuroretina cells.

We previously reported that avian retroviruses carrying the v-myc oncogene alone fail to induce sustained proliferation and transformation of non-dividing chicken neuroretina (CNR) cells from 7-day-old embryos. However, v-myc is capable of transforming CNR cells which have been induced to multiply by the v-mil oncogene. These results suggest that entry into the cell cycle is required for the transformation of CNR cells by v-myc. To further assess the role of cell division, we investigated the transforming properties of v-myc in CNR cells conditionally induced to divide by the v-src gene or by modified culture conditions. We show that v-myc transforms CNR cells infected with Rous sarcoma virus mutants which induce cell proliferation in the absence of transformation. Expression of these transforming properties in CNR cells infected with temperature-sensitive v-src mutants depends on the continuous mitogenic activity of p60v-src. We also report that v-myc is able to transform CNR cells and to increase their growth potential under culture conditions which allow transient multiplication of uninfected cells. However, these v-myc-transformed cells rapidly cease to divide when returned to culture conditions that restrict the growth of normal cells. Taken together, these results indicate that transformation of CNR cells by the v-myc oncogene continuously depends on their ability to enter the cell cycle.

Animals↗
PubMed1990

Transformation-defective mutants with 5' deletions of the src gene are frequently generated during replication of Rous sarcoma virus in established quail fibroblasts.

Replication of Rous sarcoma virus (RSV) in avian fibroblasts leads to the generation of replication-competent variants that are defective for cell transformation (td virus). These td variants contain deletions affecting various portions of the v-src gene. We compared the rate of td virus production in Q3B cells, a quail cell line established by mutagen treatment, and in normal quail fibroblasts. Twenty-five days after infection with an RSV stock containing only transforming virions, Q3B cells harbor similar amounts of v-src-containing and v-src-deleted proviruses. However, these cells synthesize very low levels of p60v-src and generate large excess of td variants, as determined by biological assays. Unlike Q3B cells, normal quail fibroblasts infected with the same virus stock produce td variants only after multiple passages of undiluted virus on fresh cells. Restriction analysis showed that the td virus produced by Q3B cells is composed of two types of genomes: one lacking the entire v-src gene and the other carrying partial deletions of this gene predominantly located in the amino-terminal portion of the coding region of v-src. To study the mechanisms of these partial deletions, we molecularly cloned and sequenced the v-src genes of several td proviruses. We show that these mutants carry single or multiple v-src deletions of limited size, presumably generated by multiple mechanisms. Two deletions of 170 and 112 bp located in the 5' portion of v-src are frequently generated during RSV replication in Q3B cells and may represent preferential sites for v-src deletion in these cells.

Animals↗
PubMed1990

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

Animals↗
PubMed1990

Down regulation by p60v-src of genes specifically expressed and developmentally regulated in postmitotic quail neuroretina cells.

The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation.

Animals↗
PubMed1989

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Amino Acid Sequence↗
PubMed1989

Expression of a novel gene encoding a 51.5 kD precursor protein is induced by different retroviral oncogenes in quail neuroretinal cells.

A cDNA clone, named T64, was isolated from a library of quail neuroretinal cells transformed by a thermosensitive v-src mutant of Rous sarcoma virus. it corresponds to the most abundant mRNA with thermodependent expression in these cells. T64 accumulation also correlated with pp60v-src activity in other cell types transformed by RSV, such as fibroblasts and myoblasts, but was independent of the proliferative state of the cells, indicating that T64 is rather implicated in the process of morphological transformation. Nuclear run on experiments showed that the accumulation of T64 mRNA in transformed neuroretinal cells is the consequence of an increased transcription rate. Enhancement of T64 expression on QNR cells was also achieved by infection with avian retroviruses harboring other oncogenes with protein kinase activity such as v-fps and v-mil. The 1.6 kb T64 mRNA was detected in vivo in a few quail tissues at levels 50-200-fold lower than in RSV-infected cells. DNA sequencing of the T64 cDNA revealed an open reading frame encoding a 449 amino acids protein with a typical N-terminal signal peptide and with significant amino acid sequence homology with a rat-secreted protein.

Amino Acid Sequence↗
PubMed1988

A novel oncogene related to c-mil is transduced in chicken neuroretina cells induced to proliferate by infection with an avian lymphomatosis virus.

Non-dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV-1. This oncogene, designated v-Rmil, is 70.1% homologous to v-mil. v-Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v-mil protein. Divergence with the v-mil gene product is observed at the NH2- and COOH-terminal portions of the v-Rmil protein. Restriction analysis of normal chicken DNA indicated that v-Rmil is derived from a cellular gene distinct from c-mil. The c-Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Amino Acid Sequence↗
PubMed1988

Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

Animals↗
PubMed1988

Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.

Animals↗