5' viral and human cellular sequences corresponding to the transforming gene of simian sarcoma virus

The 5' nucleotide sequences of the transforming gene of simian sarcoma virus (v-sis) and its human cellular homolog (c-sis) were compared. A short homology was found between helper virus and cellular DNA sequences at the junction of v-sis and c-sis, which may have had a role in the original recombination event leading to the generation of simian sarcoma virus.     

Human-proto-oncogene nucleotide sequences corresponding to the transforming region of simian sarcoma virus

The nucleotide sequences of the six regions within the normal human cellular locus (c-sis) that correspond to the entire transforming region of the simian sarcoma virus (SSV) genome (v-sis) were determined. The regions are bounded by acceptor and donor splice sites and, except for region 6, resemble exons. Region 6 lacks a 3' donor splice site and terminates -5 base pairs from the 3' v-sis-helper-viral junction. This is consistent with a model proposing that SSV was generated by recombination between proviral DNA of a simian sarcoma associated virus and proto-sis and that introns were spliced out subsequently from a fused viral-sis messenger RNA. This also suggests that the 3' recombination occurred within an exon of the woolly monkey (Lagothrix) genome. The open reading frames predicting the v-sis and c-sis gene products coincide with the stop codon of c-sis located 123 nucleotides into the fifth region of homology. The overall nucleotide homology was 91 percent with substitutions mainly in the third codon positions within the open reading frame and with greatest divergence within the untranslated 3' portion of the sequences. The predicted protein products for v-sis and c-sis are 93 percent homologous. The predicted c-sis gene product is identical in 31 of 31 amino acids to one of the published sequences of platelet-derived growth factor. Thus, c-sis encodes one chain of human platelet-derived growth factor.     

The role of individual cysteine residues in the structure and function of the v-sis gene product

The v-sis oncogene encodes a platelet-derived growth factor (PDGF)-related product whose transforming activity is mediated by its functional interaction with the PDGF receptor. PDGF, as well as processed forms of the v-sis gene product, is a disulfide-linked dimer with eight conserved cysteine residues in the minimum region necessary for biologic activity. Site-directed mutagenesis of the v-sis gene revealed that each conserved cysteine residue was required directly or indirectly for disulfide-linked dimer formation. However, substitution of serine for cysteine codons at any of four positions had no detrimental effect on transforming activity of the encoded v-sis protein. These results establish that interchain disulfide bonds are not essential in order for this protein to act as a functional ligand for the PDGF receptor. The remaining four substitutions of serine for cysteine each inactivated transforming function of the molecule. In each case this was associated with loss of a conformation shown to involve intramolecular disulfide bonds. These studies provide insight into the role of individual cysteine residues in determining the structure of the sis/PDGF molecule critical for biological activity.     

Transformation by v-sis occurs by an internal autoactivation mechanism

Transformation by the v-sis oncogene appears to require an interaction of its protein product, p28v-sis, with the receptor for the platelet-derived growth factor (PDGF). However, this interaction may not occur at the cell surface as predicted by the autocrine hypothesis because phenotypic transformation was not reversed by incubation of SSV-NRK cells with antisera to PDGF and because morphological transformation did not occur when nontransformed NRK cells were cultured continuously with p28v-sis. A mutant of the wild-type v-sis gene was constructed that encodes a v-sis protein targeted for retention within the endoplasmic reticulum and Golgi. NRK cells expressing the mutant v-sis gene did not secrete any detectable v-sis protein but were as fully transformed as wild-type v-sis transfectants. The results support a mechanism of transformation by v-sis in which internal activation of the PDGF receptor occurs before expression of either p28v-sis or the PDGF receptor at the cell surface.     

Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor

A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors.     

Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains

Human platelet-derived growth factor (PDGF) consists of two distinct but related polypeptide chains designated PDGF-A and PDGF-B. The gene encoding PDGF-B has given rise to the v-sis oncogene. In the present study the transforming activities of PDGF-A and PDGF-B genes are compared. The PDGF-A chain gene is markedly less efficient in inducing transformation than the PDGF-B gene under the influence of the same promoter. There are significant differences in the secretory and growth stimulating properties of the two chains. These properties appear to account for the much more potent transforming ability of the PDGF-B gene. These findings provide insights into biologic properties of a growth factor responsible for potent autocrine stimulation of abnormal cell proliferation.     

Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells

Autocrine activation of platelet-derived growth factor (PDGF) receptors is the mechanism of transformation by the v-sis oncogene. Since the addition of PDGF does not transform normal cells, autocrine mechanisms may involve unique pathways of receptor activation. In this study autocrine stimulation of the PDGF receptor was observed in v-sis-transformed normal rat kidney (NRK) cells. In contrast to receptor activation in normal cells, autocrine activation of PDGF receptors in v-sis-transformed cells occurred in intracellular compartments, disrupting receptor processing and diverting receptors and their precursors to a chloroquine-sensitive degradation pathway. These findings show that intracellular activation of receptors by autocrine mechanisms may play a role in cell transformation.     

Chromosomal localization of the human homolog (c-sis) of the simian sarcoma virus onc gene

Nonrandom chromosome rearrangements of chromosome 22 have been identified in different human malignancies. As a result of Southern blot hybridization of a c-sis probe to DNA's from mouse-human somatic cell hybrids, the human homolog (c-sis) of the transforming gene of simian sarcoma virus was assigned to chromosome 22. Hybrids between thymidine kinase-deficient mouse cells and human fibroblasts carrying a translocation of the region q11-qter of chromosome 22 to chromosome 17 were also analyzed. These studies demonstrate that the human c-sis gene is on region 22q11 greater than qter.     

Molecular localization of the transforming and secretory properties of PDGF A and PDGF B

Human platelet-derived growth factor (PDGF) is a connective tissue cell mitogen comprised of two related chains encoded by distinct genes. The B chain is the homolog of the v-sis oncogene product. Properties that distinguish these ligands include greater transforming potency of the B chain and more efficient secretion of the A chain. By a strategy involving the generation of PDGF A and B chimeras, these properties were mapped to distinct domains of the respective molecules. Increased transforming efficiency segregated with the ability to activate both alpha and beta PDGF receptors. These findings genetically map PDGF B residues 105 to 144 as responsible for conformational alterations critical to beta PDGF receptor interaction and provide a mechanistic basis for the greater transforming potency of the PDGF B chain.     

Vascular permeability factor, an endothelial cell mitogen related to PDGF

Vascular permeability factor (VPF) is a 40-kilodalton disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth, and angiogenesis. These properties suggest that the expression of VPF by tumor cells could contribute to the increased neovascularization and vessel permeability that are associated with tumor vasculature. The cDNA sequence of VPF from human U937 cells was shown to code for a 189-amino acid polypeptide that is similar in structure to the B chain of platelet-derived growth factor (PDGF-B) and other PDGF-B-related proteins. The overall identity with PDGF-B is 18%. However, all eight of the cysteines in PDGF-B were found to be conserved in human VPF, an indication that the folding of the two proteins is probably similar. Clusters of basic amino acids in the COOH-terminal halves of human VPF and PDGF-B are also prevalent. Thus, VPF appears to be related to the PDGF/v-sis family of proteins.     

Genetic mapping of the mouse proto-oncogene c-sis to chromosome 15

The mouse homolog (c-sis) of the transforming gene of the simian sarcoma virus was mapped to chromosome 15 by the Southern blot analysis of DNA's from hamster-mouse somatic cell hybrids. Alterations in c-sis expression may thus play a role in the various murine neoplastic diseases characterized by rearrangements or duplications of chromosome 15.     

Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor

Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived growth factor (PDGF). Like PDGF, the growth factor derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human fibroblasts. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human fibroblasts. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of 17 kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.     

PDGF及其受体mRNA在鹅不同等级前卵泡中的表达差异

为探讨血小板衍生生长因子(Platelet-derived growth factor,PDGF)及其受体(PDGFR)mRNA在鹅等级前卵泡中的表达差异及生物学意义。选取健康的产蛋前期和产蛋期籽鹅各3只为试验材料,利用实时荧光定量PCR方法检测PDGF和PDGFR mRNA在产蛋前期和产蛋期鹅等级前卵泡(分为SYF、LWF、MWF、SWF、PE 5个等级)中的表达差异。结果表明:产蛋期PDGF及其受体mRNA在5个等级前卵泡中的表达量均高于产蛋前期(P0.05),说明PDGF及其受体mRNA对卵泡发育有促进作用;产蛋前期和产蛋期PDGF及其受体mRNA表达量均在初级卵泡(PE)中最高,说明初级卵泡是主要表达部位,其中颗粒细胞形态由扁平变成立方形后开始大量增殖,通过促进颗粒细胞的增殖进而促进卵泡发育;在初级卵泡向小白卵泡(SWF)发育过程中,PDGF及其受体mRNA表达量有明显下降(P0.05),推测这可能与PDGF抑制类固醇的合成进而抑制卵泡发育有关,使优势卵泡更有序的进行筛选。     

Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA

Both interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) induce proliferation of cultured fibroblasts and smooth muscle cells. These polypeptide mediators are released by activated macrophages and other cell types in response to injury and are thought to have a role in tissue remodeling and a number of pathologic processes. Analysis of the kinetics of [3H]thymidine incorporation by cultured fibroblasts demonstrated that the response to IL-1 is delayed approximately 8 hours relative to their response to PDGF. IL-1 transiently stimulated expression of the PDGF A-chain gene, with maximum induction after approximately 2 hours. Subsequent synthesis and release of PDGF activity into the medium was detected as early as 4 hours after IL-1 stimulation, and downregulation of the binding site for the PDGF-AA isoform of PDGF followed PDGF-AA secretion. Antibodies to PDGF completely block the mitogenic response to IL-1. Therefore, the mitogenic activity of IL-1 for fibroblasts and smooth muscle cells appears to be indirect and mediated by induction of the PDGF A-chain gene.     

Vasoconstriction: a new activity for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.     

Two classes of PDGF receptor recognize different isoforms of PDGF

Previous studies involving platelet-derived growth factor (PDGF) have been based on the premise that a single cell-surface receptor binds all three isoforms of PDGF (AA, BB, and AB). It is now shown that two populations of PDGF receptor exist and can be distinguished by their ligand binding specificity. The B receptor binds only the BB dimer, whereas the A/B receptor binds AA, BB, and AB dimers. Human dermal fibroblasts appear to express seven times as much B receptor as A/B receptor. The B receptor is responsible for most PDGF receptor phosphorylation.     

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.     

产Zwittermicin A的苏云金芽孢杆菌菌株的筛选及抗性基因zmaR的克隆与表达

 通过PCR方法筛选160株苏云金芽孢杆菌，其中94株含有zmaR基因。测定了含zmaR基因的菌株培养物上清液对草生欧文氏杆菌（Erwinia herbicola）的抑菌活性，结果表明有67株菌有抑菌活性，其中21株抑菌活性较高。经鉴定，抑菌活性较高的G03菌株含有cry1Ac、cry1Aa、cry1Ca和cry2Ab等高毒力杀虫基因，并从G03中克隆了zmaR全长基因，完成了序列测定。该基因编码区为1 125 bp，由核苷酸序列推导的氨基酸残基组成为375个，分子量为43.5 kD，等电点pI4.945。通过载体pET-21b将zmaR基因导入大肠杆菌BL21，可正常表达43.5 kD蛋白，并使宿主菌产生对Zwittermicin A的抗性。     

Role of phosphatidylinositol kinase in PDGF receptor signal transduction

The molecules with which the platelet-derived growth factor (PDGF) receptor interacts to elicit the biochemical reactions responsible for cell proliferation have not been identified. Antisera directed against specific PDGF receptor peptides coprecipitated a phosphatidylinositol (PI) kinase and the PDGF receptor. Immunoprecipitates from PDGF-stimulated cells contained 10 to 50 times as much PI kinase as those from unstimulated cells. Mutation of the PDGF receptor by deletion of its kinase insert region resulted in a receptor markedly less effective than the wild type in eliciting cell proliferation and defective in PDGF-stimulated PI kinase, but still capable of PDGF-induced receptor autophosphorylation and phosphoinositide hydrolysis. These data show that the PDGF receptor is physically associated with a PDGF-sensitive PI kinase that is distinct from tyrosine kinase and is not required for PDGF-induced PI hydrolysis. The finding that the mutant PDGF receptor missing the kinase insert domain elicited known early biochemical responses to PDGF, but did not associate with or regulate PI kinase, suggests a novel role for the receptor-associated PI kinase in the transmission of mitogenic signals.