Bawden C S, Sivaprasad A V, Verma P J, Walker S K, Rogers G E
Department of Biochemistry, University of Adelaide, South Australia.
Transgenic Res. 1995 Mar;4(2):87-104. doi: 10.1007/BF01969411.
It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesise cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes of Salmonella typhimurium, cysE, cysM and cysK, and linked cysEM, cysME and cysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and -cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and -cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cells in vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.
通过增加毛囊中可用于蛋白质合成和细胞分裂的半胱氨酸供应量，有可能提高羊毛产量。由于哺乳动物只能通过转硫化作用从蛋氨酸间接合成半胱氨酸，因此编码微生物半胱氨酸生物合成酶的转基因表达可为绵羊的半胱氨酸合成提供更有效的途径。如果在瘤胃上皮中表达，瘤胃微生物产生并通常排出的大量硫化物可被捕获用于转化为半胱氨酸。本文描述了鼠伤寒沙门氏菌半胱氨酸生物合成基因cysE、cysM和cysK以及相连的cysEM、cysME和cysKE基因作为转基因在小鼠和绵羊中的表达特征。相连的转基因构建时每个基因由单独的启动子驱动，该启动子可以是劳氏肉瘤病毒长末端重复序列（RSVLTR）启动子或小鼠磷酸甘油酸激酶-1（mPgk-1）启动子，并带有人生长激素（hGH）聚腺苷酸化序列。用RSVLTR-cysE基因对小鼠进行转基因可实现该基因的组织特异性、可遗传表达。尽管在许多组织中表达水平很高，但在胃和小肠中的表达水平极低。使用RSVLTR-cysEM和-cysME相连转基因进行的同期绵羊转基因实验结果表明，RSVLTR启动子不足以在瘤胃中表达。此外，观察到含有RSVLTR序列的转基因不稳定。mPgk-cysME和-cysKE相连转基因在包括胃和小肠在内的大多数被检测小鼠组织中的表达表明，该启动子更适合在绵羊的类似组织中表达这些转基因。然而，随后的绵羊转基因实验表明，在发育早期普遍活跃的mPgk-1启动子可能不适用于半胱氨酸生物合成转基因的表达。总之，这些结果表明，具有酶活性的细菌半胱氨酸生物合成基因产物可以在体内哺乳动物细胞中共表达，但基因表达应在时空上局限于成年绵羊瘤胃上皮。
