基因工程与生命科学

一、基因工程的兴起与发展 在分子生物学和分子遗传学的发展史上出现过许多惊人的成就,它们对基因工程的诞生直接或间接的起到了推波助澜的作用。在众多的成就之中,我们将对基因工程诞生影响最大的概括为理论上的三大发现和技术上的三大发明。 1944年,Avery通过肺炎球菌转化实验指出,生物遗传性的物质基础是DNA。     

肺炎双球菌的转化现象

肺炎双球菌的转化现象是1928年由格里芬(Griffith)所作的肺炎双球菌对小家鼠的感染实验中发现的。十六年后,阿维利(Avery)等重复了上述实验,并进一步将有荚膜肺炎双球菌的DNA、蛋白质、荚膜提纯,然后分别与活的无荚膜的肺炎双球菌混合,结果证实能起转化作用的是DNA,而蛋白质、荚膜等物质并无作用。     

真核生物的基因组织和结构

真核生物的基因组DNA被包装在染色体中。染色体的化学物质是染色质,它是由DNA和核蛋白组成。染色体DNA含有几乎所有真核生物的基因、重复序列和基因间序列。基因是由一段DNA序列组成,有编码序列和非编码序列。真核基因的编码序列是不连续的,它被插入序列分隔,而各真核基因之间则由基因间序列相隔。基因的结构和表达与核内蛋白质有着密切的关系。     

肽核酸的特性及作用

肽核酸（peptide nucleic acid,PNA)是一类DNA结构类似物,能与DNA和RNA特异性杂交。它通过与DNA结合而抑制基因转录,通过与mRNA结合而阻止蛋白质的合成;同时,PNA不易被核酸酶和蛋白酶降解,所有这些显示出其作为反基因药物和反义药物的良好应用前景。     

基因的转录都必须是连续的吗?

蛋白质的一级结构是由 mRNA 决定的,而 mRNA 是在基因组的一段 DNA 上合成的。通常认为一段连续不断的 DNA 为一分子蛋白质的编码,亦即 mRNA 是直接从这一段 DNA的顺序连接地转录下来的。这段 DNA 就是这个要被翻译出的蛋白质的基因。原核生物方面的实验无疑是支持这种看法的,不过真核生物方面的一些实验结果却很不相同。     

“垃圾”基因，垃圾还是宝物？

何谓“垃圾”基因 “垃圾”基因是相对基因而言的。基因是具有遗传效应的特定DNA序列,通俗地讲,基因就是编码某种蛋白质的一段基因。它们就像散落于天幕的星星一样,分散在我们的基因组中。而在这些基因间存在的大片大片的DNA片段是不能编码蛋白质的,即“非编码序列”。由于功能不清,美国加州理工学院的大野·乾于1972年提出用“垃圾基因”（junk DNA）来形容它们。     

理查德·约翰·罗伯茨

1944年,艾弗里通过肺炎双球菌转化实验证明了DNA是遗传物质,从而使人们对基因的认识有了根本性改变.在20世纪70年代,尽管转座子的存在已被证明,但大多数科学家都认为基因是连续排列在DNA上的一段有功能的片段(转座子插入破坏了这种完整性而使基因失活),然而1977年两位科学家"基因内不编码序列(内含子)"的独立发现使传统的基因概念发生了动摇,大大推动了分子生物学的迅猛发展.两位科学家之一就是美国新英格兰生物实验室的理查德·约翰·罗伯茨(Richard John Roberts).     

白喉毒素A链基因在植物中的表达能抑制蛋白质合成

利用原生质体瞬间表达系统证实,即使用大量蓖麻毒素A链基因DNA导入诸葛莱(Ory-chophoragmus violaceus)原生质体,其蛋白质合成仍不被抑制。但是诸葛菜和灰叶烟草(Nicotiana plumbagenifolia)原生质体的蛋白质合成却容易被表达的白喉毒素A链所抑制。用CaMV 35 S启动子和Kozak序列控制的白喉毒素A链嵌合基因DNA在3h内能完全中止灰叶烟草原生质体蛋白质合成,此期间蛋白合成总量仅为正常时5%左右。     

细菌遗传物质的结构及在遗传工程中的作用

在电镜下观察,细菌细胞中不存在真正的核,DNA被局限在细胞的中心部位,它被细胞的其他部分所包围。与真核细胞相比,这个局限细菌DNA的区域常常被称为细菌的“核”;而细胞的其余部分常常被称为细菌的“细胞质”。在这种核与细胞质之间,不存在使二者隔离的膜,即细菌没有核膜。而细菌的DNA,尽管并没有象真核细胞的DNA那样与蛋白质结合,也常常被称为细菌的“染色体”,其分子量大约是1～6×10~9道尔顿。在这种染色体上,排列着细菌遗传的全部或大部分基因,它是细菌赖以生存的基本遗传物质。     

细胞核的功能

真核细胞的显著特点之一是具有膜包裹着的细胞核,其主要功能是将基因的转录和信使核糖核酸指导合成蛋白质这2个过程在空间上分隔开。最初为蛋白质编码的RNA序列可能是被不为蛋白质编码的RNA序列所隔断的,这些非编码区后来成为DNA分子中基因里的内含子。原核细胞为了节省资源,已基本清除了基因中的内含子。而真核细胞则利用内含子,用同一个基因合成多种蛋白质,这就需要细胞核阻止含有内含子的m RNA与合成蛋白质的核糖体接触。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Scurvy in capybaras bred in captivity in Argentine

In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.     

Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.