鸡卵清蛋白基因第一内含子和3′-调控区对外源基因表达的调控作用

探明鸡卵清蛋白基因(ov)第一内含子和 3'-调控区对目的基因表达水平的影响,为鸡输卵管表达载体的构建提供科学依据.用牛生长激素基因(BGH)poly A 序列替换鸡输卵管表达载体中 ov 3'-调控区,用限制酶消化法逐步删减第一内含子序列,获得5个表达人组织激肽释放(hK1)cDNA 的鸡输卵管表达载体,分别命名为 pOV2K、pOV3K、pOV4K、pOV5K 和 pOV6K.经翅静脉给产蛋鸡注射聚乙烯亚胺包裹的相同拷贝数重组载体,通过蛋清中酶活性定量检测评价不同载体的表达水平.结果表明:受控于 3.0kb ov 5'-和3'-调控区的 pOV2K 表达的重组酶活性明显高于用 BGH poly A 替换 3'-调控区的pOV3K;无内含子的pOV6K表达水平显著低于舍不同长度第一内含子的pOV3K、pOV4K 和 pOV5K,重组酶表达水平随第一内舍子的缩短呈逐渐下降趋势.该试验结果表明 ov 第一内含子和 3'-调控区对目的基因在鸡榆卵管细胞中的表达具有正调节作用,应当包括在鸡输卵管表达载体中.     

Afferent input from rat slow skeletal muscle inhibits bioassayable growth hormone release.

The release of a bioassayable form of growth hormone (BGH), distinct from growth hormone as measured by immunoassay (IGH), from the rat pituitary into the blood is differentially regulated by afferent input from fast and slow skeletal muscles. Specifically, activation of low-threshold fast muscle afferents for 15 min increased plasma BGH by 217 and 295% and decreased pituitary BGH by 68 and 45% in male and female rats, respectively. In contrast, activation of slow muscle afferents inhibited BGH release, decreasing plasma BGH by approximately 60% and increasing pituitary BGH by 30-50% in male rats. Female rats from which food had been withheld for approximately 12 h had elevated basal plasma BGH levels, which then were decreased by 81% after slow muscle nerve stimulation. Plasma IGH concentrations were unchanged after any nerve stimulation condition. These results demonstrate that regulation of BGH release can be differentially mediated through low-threshold afferent inputs from fast or slow skeletal muscle. Furthermore, the results indicate that BGH responses are independent of gender or feeding status.     

Basal and evoked levels of bioassayable growth hormone are altered by hindlimb unloading.

Bioassayable growth hormone (BGH) in rats is released in large quantities from the pituitary in response to the activation of large, proprioceptive afferent fibers from fast and mixed fiber-type hindlimb musculature. We hypothesized that hindlimb unloading (HU) of adult male rats would 1) reduce the basal levels of plasma BGH, and 2) abolish stimulus-induced BGH release. Rats were exposed to HU for 1, 4, or 8 wk. Plasma and pituitaries were collected under isoflurane anesthesia for hormone analyses. Additionally, at 4 and 8 wk, a subset of rats underwent an in situ electrical stimulation (Stim) of tibial nerve proprioceptive afferents. Basal plasma BGH levels were significantly reduced (-51 and -23%) after 1 and 8 wk of HU compared with ambulatory controls (Amb). Although Amb-Stim rats exhibited increased plasma BGH levels (88 and 143%) and decreased pituitary BGH levels (-27 and -22%) at 4 and 8 wk, respectively, stimulation in HU rats had the opposite effect, reducing plasma BGH (-25 and -33%) and increasing pituitary BGH levels (47 and 10%) relative to HU alone at 4 and 8 wk. The 22-kDa form of GH measured by immunoassay and the plasma corticosterone, T3, T4, and testosterone levels were unchanged by HU or Stim at all time points. These data suggest that BGH synthesis and release from the pituitary are sensitive both to chronically reduced neuromuscular loading and to acute changes in neuromuscular activation, independent of changes in other circulating hormones. Thus BGH may play a role in muscle, bone, and metabolic adaptations that occur in response to chronically unloaded states.     

Cloning of bovine growth hormone gene and its expression in bacteria.

A hybrid plasmid was constructed containing beta-lactamase gene of plasmid pBR322 and cloned coding sequences of bovine growth hormone (BGH). The constructed plasmid contains all DNA sequences required to encode BGH, and when used as a hybridization probe it detects one growth hormone gene in the bovine genome. The cloned DNA sequences are inserted into the beta-lactamase gene in the correct reading frame for BGH synthesis. The hybrid gene is expressed in bacteria and the product, a fused beta-lactamase-bovine growth hormone protein, is specifically immunoprecipitated with anti-serum to BGH. Unlike beta-lactamase, very little growth hormone containing sequences can be detected in the periplasmic space.     

Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.     

Bioassayable growth hormone release in rats in response to a single bout of treadmill exercise.

Plasma growth hormone (GH) measured by immunoassay [immunoassayable GH (IGH)] and by tibial bioassay [bioassayable GH (BGH)] increases in humans in response to exercise. In rats, however, IGH does not change in response to exercise. The objective of this study was to determine the BGH response to an acute exercise bout in rats. The rats ran on a treadmill at a rate of 27 m/min for 15 min, after which plasma and pituitary hormones, including IGH and BGH, and plasma metabolites were measured. Plasma and pituitary IGH were unchanged from control groups after the acute exercise bout, whereas plasma BGH was increased by 300% and pituitary BGH was decreased by 50%. Plasma thyroxine and corticosterone levels were significantly increased after a single exercise bout, but plasma testosterone, 3,5, 3'-triiodothyronine, glucose, lactate, and triglyceride concentrations were unchanged. Given previous results from in situ nerve stimulation studies (Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, Grossman EJ, and Edgerton VR. J Appl Physiol 84: 1425-1430, 1998), these in vivo results are consistent with the rapid BGH response during exercise being induced by the activation of muscle afferents.     

Spaceflight suppresses exercise-induced release of bioassayable growth hormone.

We have reported that bed rest suppressed the release of bioassayable growth hormone (BGH) that normally occurs after an acute bout of unilateral plantar flexor exercise (G. E. McCall, C. Goulet, R. E. Grindeland, J. A. Hodgson, A. J. Bigbee, and V. R. Edgerton. J. Appl. Physiol. 83: 2086-2090, 1997). In the present study, the effects of spaceflight on the hormonal responses to this exercise protocol were examined. Four male astronauts on the National Aeronautics and Space Administration Shuttle Transport System (STS-78) mission completed the exercise protocol before, during, and after a 17-day spaceflight. The maximal voluntary contraction torque output at the onset of exercise was similar on all test days. Before spaceflight, plasma BGH increased 114-168% from pre- to postexercise. During spaceflight and after 2 days recovery at normal gravity (1 G), the BGH response to exercise was absent. After 4 days of recovery, this response was restored. Plasma concentrations of immunoassayable growth hormone were similar at all time points. The preexercise plasma immunoassayable insulin-like growth factor I (IGF-I) levels were elevated after 12 or 13 days of microgravity, and a approximately 7% postexercise IGF-I increase was independent of this spaceflight effect. The suppression of the BGH response to exercise during spaceflight indicates that some minimum level of chronic neuromuscular activity and/or loading is necessary to maintain a normal exercise-induced BGH release. Moreover, these results suggest that there is a muscle afferent-pituitary axis that can modulate BGH release.     

Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I

Linear expression constructs can facilitate gene function studies. We describe a method to generate linear expression constructs for mammalian cells by one-step polymerase chain reaction (PCR) with vaccinia DNA topoisomerase I (TOPO). Cytomegalovirus (CMV) 5′ promoter, the gene of interest, and V5 bovine growth hormone (BGH) polyA 3′ terminator elements were PCR-amplified with target-specific primers containing vaccinia DNA TOPO-specific sequence and complementary sequence to each other. We amplified specific and complementary sequences. These three elements were directionally joined with vaccinia TOPO. The joined products were then directly transfected into Chinese hamster ovary cells. Compared with the transfection of supercoiled plasmids, comparable expression signals were obtained for green fluorescent protein, chloramphenicol acetyltransferase, and β-galactosidase proteins using Western blots. This is a quick and efficient method to generate linear expression construct. Unlike Invitrogen TOPO Tools, our method avoided the secondary round of PCR and more rapidly yielded correct joining products. This method can be easily used in the function test of uncharacterized open reading frames.     

The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases

BACKGROUND: Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease-rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease-resistant a priori. METHODS AND RESULTS: The hypothesis that a significant part of nuclease attack is directed towards certain labile sequences in a pDNA model (pVAX1/lacZ) was first tested. Homopurine-rich tracts in the bovine growth hormone polyadenylation signal (BGH poly A) were identified as labile sequences using S1 nuclease as a probe. Two pDNA variants were then created by replacing the BGH poly A region with the SV40 or a synthetic poly A signal. A study of plasmid degradation in eukaryotic cell lysates and mice plasma showed that the half-life of the supercoiled isoforms of the new vectors was always higher when compared with the control plasmid. An in vitro assay of the reporter beta-galactosidase in transfected CHO cells further showed that gene expression with the new pDNA variants was not affected negatively by the plasmid modifications. CONCLUSIONS: The replacement of labile sequences in plasmid DNA vectors improves resistance towards nuclease attack as shown by the increased half-lives of supercoiled plasmid isoforms incubated with endo/lysosomal, cytoplasmatic and blood plasma enzymes.     

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.     

在哺乳动物细胞中稳定表达丙型肝炎病毒E2糖蛋白

利用ＤＮＡ重组技术,将Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段插入真核表达载体,然后转染哺乳动物细胞ＮＩＨ３Ｔ３以表达Ｅ２糖蛋白．检测显示来自３月以上培养的细胞克隆中表达产物分子量为７０ｋＤ,经Ｗｅｓｔｅｒｎｂｌｏｔ证实该表达产物能与抗ＨＣＶ阳性血清进行特异性反应．以上表明首次在哺乳动物细胞中成功表达Ⅲ型中国株ＨＣＶ的Ｅ２糖蛋白,并建立相应的稳定表达细胞系．     

Influences of stress, age and sex on serum growth hormone and free fatty acid levels in cattle.

The serum growth hormone (BGH) and free fatty acid (FFA) concentrations of large groups of calves (male and female ranging in age from 8 to 14 days and from 3 to 6 months old), bulls, oxen, heifers and cows under normal conditions were compared with the serum BGH and FFA values of corresponding large groups of animals under stress conditions. Stress was caused by transport, as it routinely occurs, and was further reinforced by a stay of several hours in unfamiliar surroundings. Stress provokes a significant increase in serum FFA in all groups and induced a significant decrease in serum BGH in all groups, with the exception of the non-pregnant heifers. The male and female calves which were 8 to 14 days old, had significantly higher BGH levels than the older animals. The female calves from 3 to 6 months of age, and the heifers had significantly lower BGH levels than the cows. The males had higher serum BGH values than the females, but a significant difference could be demonstrated only between the male and female calves of both age-groups. The oxen, which were 1 to 3 years old, had significantly higher BGH levels than the non-pregnant heifers of the same age. It appeared that gestation had no influence on the serum BGH and FFA levels of both heifers and cows.     

Muscle afferent activity modulates bioassayable growth hormone in human plasma.

Immunoassayable and bioassayable growth hormone responses to vibration-induced activation of muscle spindle afferents of the soleus (Sol) or tibialis anterior (TA) muscles were studied in 10 men. Subjects were supine while a 10-min vibration stimulus (100 Hz; 1.5-mm amplitude) was applied to the muscle, with each of the muscles tested on separate days. Blood samples were collected before, during, immediately after, and after 5 and 10 min of vibration. Plasma growth hormone concentrations were determined by radioimmunoassay (IGH) for all sampling periods and by bioassay (BGH; measurement of tibial epiphysial cartilage growth in hypophysectomized rats) for samples obtained before and immediately after vibration. Plasma IGH concentrations were similar at all time points during the Sol or TA experiments. After 10 min of muscle vibration, mean plasma BGH was elevated 94% [1,216 +/- 148 (SD) to 2, 362 +/- 487 microg/l; P = 0.0001] for TA and decreased 22% (1,358 +/- 155 to 1,058 +/- 311 microg/l; P = 0.09) for Sol. These data demonstrate that activation of TA muscle spindle afferents increases circulating BGH but not IGH. The absence of a similar vibration-induced BGH response for the Sol indicates a differential regulation of BGH release by these two predominantly slow muscles, perhaps related to their respective flexor and extensor functions. These data indicate that a muscle afferent-pituitary axis modulates the release of BGH, but not IGH, from the pituitary in humans and that this axis is muscle specific, similar to that observed in rats.     

Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.     

新型双基因表达盒真核载体的构建及功能验证

目的: 构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,并对其功能进行验证。方法: 设计一个包含人巨细胞病毒启动子(CMV promoter)、T7启动子、信号肽基因、血凝素A表位基因(HA)、多克隆位点区域(MCS)、c-myc抗原表位、血小板来源的生长因子受体跨膜区域(PDGFR TM)及牛生长激素多腺苷酸化信号(BGH polyA)等八种元素的表达盒Ⅱ(Expressing cassette Ⅱ),在其上下游添加Nru Ⅰ酶切位点后,进行人工化学合成,连接到克隆载体pGH中得pGH-Ⅱ;用Nru Ⅰ酶切pGH-Ⅱ,回收1 300bp左右的片段,去磷后插入到pVAX1的相应位点,Nru Ⅰ及Bgl Ⅱ/Pst Ⅰ酶切鉴定,获得新型基因疫苗真核表达载体pVAX2;然后以增强型绿色荧光蛋白(EGFP)为报告基因,将其分别构建至两个不同的表达盒内,脂质体转染BHK-21细胞,利用RT-PCR及荧光显微镜技术进行该载体的功能验证。结果: 两个表达盒内的EGFP基因在BHK-21细胞均能高效表达,相互间不受影响,且新构建的表达盒具有蛋白展示功能。结论: 成功构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,为多价DNA疫苗的研究奠定了坚实基础。     

Vibration-induced activation of muscle afferents modulates bioassayable growth hormone release.

The effects of tendon vibration on bioassayable growth hormone (BGH) secretion from the pituitary gland were investigated in anesthetized adult male rats. The tendons from predominantly fast-twitch ankle extensor muscles (gastrocnemius and plantaris) or a predominantly slow-twitch ankle extensor (soleus) were vibrated by using a paradigm that selectively activates group Ia afferent fibers from muscle spindles. The lower hindlimb was secured with the muscles near physiological length, and the tendons were vibrated for 15 min at 150 Hz and a displacement of 1 mm. Control rats were prepared similarly, but the tendons were not vibrated. Compared with control, vibration of the tendons of the fast ankle extensors markedly increased (160%), whereas vibration of the slow soleus decreased (68%), BGH secretion. Complete denervation of the hindlimb had no independent effects on the normal resting levels of BGH, but it prevented the effects of tendon vibration on BGH secretion. The results are consistent with previous findings showing modulation of BGH release in response to in vivo activation or in situ electrical stimulation of muscle afferents (Bigbee AJ, Gosselink KL, Grindeland RE, Roy RR, Zhong H, and Edgerton VR. J Appl Physiol 89: 2174-2178, 2000; Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, and Edgerton VR. J Appl Physiol 88: 142-148, 2000; Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, Grossman EJ, and Edgerton VR. J Appl Physiol 84: 1425-1430, 1998). These data provide evidence that this previously described muscle afferent-pituitary axis is neurally mediated via group Ia afferents from peripheral skeletal muscle. Furthermore, these data show that activation of this group Ia afferent pathway from fast muscles enhances, whereas the same sensory afferent input from a slow muscle depresses, BGH release.     

Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme

The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.     

Functional limits of the araIc promoter suggest an additional regulatory site for araBAD expression

The araBAD promoter is defined, in part, by two types of cis-acting constitutive mutations, araIc at position -35 and araXc at position -10. Subcloning experiments demonstrated that the araIc and araIcXc promoters require DNA sequence information out to position -53 to -56 for maximum constitutive expression. This is 8 to 10 base pairs more DNA than is generally thought to be necessary for RNA polymerase interaction. The -53 to -56 region is required for glucose repression, suggesting that an additional factor interacts in this region and is necessary for maximum expression.