Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).     

Production of an epidermal growth factor receptor-related protein

Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, by the criteria of epidermal growth factor (EGF) binding, recognition by monoclonal and polyclonal antibodies to the EGF receptor, amino-terminal sequence analysis and carbohydrate content, is related to the cell surface domain of the EGF receptor. The high rate of production and the finding that with biosynthetic labeling the specific activity of this 105-kilodalton protein exceeds that of the intact receptor indicate that it is not derived from membrane-bound mature receptor but is separately produced by the cell. These cells thus separately synthesize an EGF receptor that is inserted into the membrane and an EGF receptor-related protein that is secreted.     

The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor

The neu oncogene, identified in ethylnitrosourea-induced rat neuroglioblastomas, had strong homology with the erbB gene that encodes the epidermal growth factor receptor. This homology was limited to the region of erbB encoding the tyrosine kinase domain. It was concluded that the neu gene is a distinct novel gene, as it is not coamplified with sequences encoding the EGF receptor in the genome of the A431 tumor line and it maps to human chromosome 17.     

Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors

A systematic series of low molecular weight protein tyrosine kinase inhibitors were synthesized; they had progressively increasing affinity over a 2500-fold range toward the substrate site of epidermal growth factor (EGF) receptor kinase domain. These compounds inhibited EGF receptor kinase activity up to three orders of magnitude more than they inhibited insulin receptor kinase, and they also effectively inhibited the EGF-dependent autophosphorylation of the receptor. The most potent compounds effectively inhibited the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on the EGF-independent proliferation of these cells. The potential use of tyrosine protein kinase inhibitors as antiproliferative agents is demonstrated.     

Chimeric NGF-EGF receptors define domains responsible for neuronal differentiation

To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction.     

A chimeric, ligand-binding v-erbB/EGF receptor retains transforming potential

Comparison of amino acid sequences from human epidermal growth factor (EGF) receptor and avian erythroblastosis virus erbB oncogene product suggests that v-erbB represents a truncated avian EGF receptor gene product. Although both proteins are transmembrane tyrosine kinases, the v-erbB protein lacks most of the extracellular ligand-binding domain and a 32-amino acid cytoplasmic sequence present in the human EGF receptor. To test the validity of the proposed origin of v-erbB and to investigate the functional significance of the deleted extracellular sequences, a chimeric gene encoding the extracellular and the transmembrane domain of the human EGF receptor joined to sequences coding for the cytoplasmic domain of the avian erbB oncogene product was constructed. When expressed in Rat1 fibroblasts, this reconstituted gene product (HER-erbB) was transported to the cell surface and bound EGF. Its autophosphorylation activity was stimulated by interaction with the ligand. Expression of the HER-erbB chimera led to anchorage-independent cell growth in soft agar and EGF-induced focus formation in Rat1 monolayers. Thus, it appears that v-erbB protein sequences in the chimeric receptor retain their transforming activity under the influence of the human extracellular EGF-binding domain.     

Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.     

Signal transduction through the EGF receptor transfected in IL-3-dependent hematopoietic cells

An expression vector for the epidermal growth factor (EGF) receptor was introduced into the 32D myeloid cell line, which is devoid of EGF receptors and absolutely dependent on interleukin-3 (IL-3) for its proliferation and survival. Expression of the EGF receptor conferred the ability to utilize EGF for transduction of a mitogenic signal. When the transfected cells were propagated in EGF, they exhibited a more mature myeloid phenotype than was observed under conditions of IL-3-directed growth. Moreover, exposure to EGF led to a rapid stimulation of phosphoinositide metabolism, while IL-3 had no detectable effect on phosphoinositide turnover either in control or EGF receptor-transfected 32D cells. Although the transfected cells exhibited high levels of functional EGF receptors, they remained nontumorigenic. In contrast, transfection of v-erbB, an amino-terminal truncated form of the EGF receptor with constitutive tyrosine kinase activity, not only abrogated the IL-3 growth factor requirement of 32D cells, but caused them to become tumorigenic in nude mice. These results show that a na?ve hematopoietic cell expresses all of the intracellular components of the EGF-signaling pathway necessary to evoke a mitogenic response and sustain continuous proliferation.     

Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor

Epidermal growth factor (EGF) treatment of A-431 epidermoid carcinoma cells elicited a redistribution of phospholipase C-gamma 1 (PLC-gamma 1) from a predominantly cytosolic localization to membrane fractions. The temporal coincidence of this redistribution with EGF stimulation of inositol phosphate formation and EGF increased phosphorylation of PLC-gamma 1 suggests that the membrane association of PLC-gamma 1 is a significant event in second messenger transduction.     

A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.

Macrophage-like U-937 cells secrete a 22-kilodalton heparin-binding growth factor that is mitogenic for BALB-3T3 fibroblasts and smooth muscle cells, but not endothelial cells. The amino acid sequence predicted from complementary DNA clones indicates that the mitogen is a new member of the epidermal growth factor (EGF) family. This heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors on A-431 epidermoid carcinoma cells and smooth muscle cells, but is a far more potent mitogen for smooth muscle cells than is EGF. HB-EGF is also expressed in cultured human macrophages and may be involved in macrophage-mediated cellular proliferation.     

鹅源草酸青霉CBHⅠ基因克隆及真核表达载体构建

【目的】鹅源草酸青霉F67(Penicillium oxalicum Currie Thom)纤维素酶二糖水解酶(cellobiohydrolase,CBHⅠ)基因克隆并构建真核表达载体,为该菌株分子特性的进一步研究及构建高效纤维素分解菌奠定基础。【方法】首先通过兼并PCR法扩增CBHⅠ基因片段,然后利用改良热不对称交错PCR(TAIL-PCR)技术克隆CBHⅠ基因5′端和3′端侧翼序列,最后采用RT-PCR法扩增鹅源草酸青霉F67CBHⅠ基因序列全长,并对该基因进行生物信息学分析,构建pPIC9K真核表达载体。【结果】分别扩增到鹅源草酸青霉F67纤维素酶CBHⅠ基因片段A(EU574736)、5′端序列B1(EU603295)、3′端序列B2(EU652768)及全长基因C(EU727171),并成功构建真核表达载体pPIC9K-CBHⅠ;生物信息学分析表明,该基因蛋白序列与微紫青霉氨基酸序列同源性最高,达76%,前26个氨基酸为信号肽序列,疏水性可达2.63,由催化功能域、衔接区和真菌性纤维素结合域构成,其三级结构主要为β-sheet。【结论】鹅源草酸青霉纤维素酶CBHⅠ基因全长序列的克隆进一步丰富了丝状真菌的生物信息学资源;其真核表达载体的构建为将该菌株进一步在真核宿主中表达,从而获得高效工程菌株奠定了基础。     

Cassette of eight exons shared by genes for LDL receptor and EGF precursor

The amino acid sequences of the human low-density lipoprotein (LDL) receptor and the human precursor for epidermal growth factor (EGF) show 33 percent identity over a stretch of 400 residues. This region of homologous is encoded by eight contiguous exons in each respective gene. Of the nine introns that separate these exons, five are located in identical positions in the two protein sequences. This finding suggests that the homologous region may have resulted from a duplication of an ancestral gene and that the two genes evolved further by recruitment of exons from other genes, which provided the specific functional domains of the LDL receptor and the EGF precursor.     

EGF、EGFR在牦牛卵母细胞中的表达及对胚胎发育能力的作用

【目的】探明表皮生长因子（epidermal growth factor,EGF）及其受体（EGFR）在牦牛卵母细胞成熟过程中的表达动态,并且研究EGF对牦牛卵母细胞成熟的影响及可能的分子作用机制。【方法】采集牦牛卵巢,捡取质量较好的牦牛卵丘-卵母细胞复合体（cumulus-oocyte complexes,COCs）,进行体外成熟培养;分别处理未成熟和成熟的牦牛COCs,采用Real-time PCR和间接免疫荧光方法检测COCs成熟过程中EGF及EGFR在基因和蛋白水平的表达动态;牦牛COCs成熟培养时在基础培养基中分别添加0、50、100和200 ng·mL^-1 EGF及最佳作用浓度的EGFR抑制因子Gefitinib,比较作用前后卵母细胞成熟率、受精后卵裂率及囊胚率;Real-time PCR方法分析不同浓度EGF和Gefitinib作用后成熟COCs中凋亡相关基因Bax和Baxi的相对表达量。【结果】EGF,EGFR基因在成熟COCs的表达显著高于未成熟COCs,成熟COCs中EGF基因的相对表达量为未成熟COCs 的2.17±0.36倍,EGFR基因在成熟COCs的表达量为未成熟COCs 6.82±0.21倍,且在未成熟和成熟COCs中EGFR基因表达量均显著高于EGF。免疫荧光检测显示未成熟COCs和成熟COCs均表达EGF和EGFR蛋白,EGF和EGFR蛋白的标记荧光主要集中在卵丘细胞,卵母细胞表达较弱。100 ng·mL^-1 EGF可以显著提高COCs成熟率（73.45±2.09）%及其受精后的卵裂率（59.46±1.41）%和囊胚率（26.23±1.08）%;对照组中（0 EGF）COCs成熟率为(54.16±3.25)%,卵裂率为 (48.33±1.93)%,囊胚率为(15.34±0.43)%;EGF浓度为50 ng·mL^-1时成熟率、卵裂率及囊胚率分别为(61.79±1.04)%、(51.76±0.61)%和(18.62±1.13)%;200 ng·mL^-1成熟率、卵裂率及囊胚率分别为(71.26±4.18)%、(57.13±2.06 )% 和(23.96±0.53)%;而加入EGFR抑制因子Gefitinib显著的降低了COCs的成熟率及其受精后的卵裂率及囊胚率,分别为(43.63±1.46)%、(41.79±2.81)%和(12.65±0.67)%。COCs成熟过程中EGF的作用可以显著抑制促凋亡基因Bax的表达,在50 ng·mL^-1、100 ng·mL^-1、200 ng·mL^-1EGF作用后成熟COCs中Bax基因的相对表达量分别仅为对照组（0 EGF）的0.83±0.12,0.21±0.02,0.27±0.03倍,EGF浓度为100 ng·mL^-1时Bax基因表达量最低,而Gefitinib作用后可以显著促进成熟COCs中Bax基因的表达,其相对表达量为对照组的4.24±0.10倍;EGF在COCs成熟过程中可以显著促进抗凋亡基因Baxi的表达,其相对表达量在EGF浓度为50、100、200 ng·mL^-1EGF分别为对照组的2.18±0.12、7.06±0.59和6.73±0.31倍,EGF浓度为100 ng·mL^-1时Baxi基因表达量最高,而Gefitinib作用后可以显著降低成熟COCs中Baxi基因的表达,其相对表达量为对照组的0.32±0.04倍。【结论】EGF和EGFR作为牦牛COCs体外成熟过程中重要的自分泌因子,且外源的EGF可以显著提高卵母细胞成熟率、卵裂率及囊胚率,最佳作用浓度为100 ng·mL^-1,其作用机制可能与调控凋亡相关基因Bax和Baxi表达有关。     

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.     

猪乳脂肪球表面因子8的克隆与表达分布

基于电子延伸序列,本试验成功克隆了猪乳脂肪球表面因子8（MFGE8）基因（GenBank登录号：EU559724）并进行了序列分析,同时利用RT-PCR方法分析了MFGE8基因的组织分布规律。序列分析结果表明MFGE8开放读码框全长为1296bp,编码431个氨基酸残基的多肽链,克隆的MFGE8基因与人、大鼠、小鼠、牛的同源性分别为71．9％、78．7％、79．1％、87．7％,推测的氨基酸序列的同源性分别为65．7％、74．0％、74．0％、84．5％。氨基酸序列N端含有Notch样“EGF”结构,C端含有FA58C结构域。构建的系统发育树显示猪与牛的亲缘关系最近。组织表达分析结果表明MFGE8在乳腺中表达最高,在大脑中表达量最低。