Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.     

羊BLG基因5′和3′调控区克隆及其调控GFP基因在乳腺细胞的表达

乳球蛋白 (ＢＬＧ)是反刍家畜的一种主要乳清蛋白质。将外源基因置于ＢＬＧ 5′调控区下游 ,能够控制外源基因在动物乳腺的组织特异性表达。现已清楚在ＢＬＧ 5′端有多个乳核因子 1(ＮＦ1)和转录因子Ｓｔａｔ5的识别位点 ,此外还存在多个激素作用位点[1 ] 。具有这些调控成分的ＢＬＧ 5′翼端 (5′ｆｌａｎｋｉｎｇ)能够控制外源基因在乳腺的表达 ,但受整合位点、外源基因结构以及转基因拷贝数及其排列方式的影响[2 ] 。研究结果表明 ,仅含有 5′调控区的乳腺表达构件 ,还不能使外源基因的表达达到内源性ＢＬＧ的表达水平 ,因为在 5′远端(…     

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.     

Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice.

The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse.     

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.     

P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.

We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.     

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.     

Deletion analysis of the human CD2 gene locus control region in transgenic mice.

Genomic sequences located at the 3' flanking region of the human CD2 gene confer high level tissue-specific, position-independent expression of the gene when introduced in the germ line of mice. In order to further characterize these sequences a range of deletions, from the 3' end were produced and transgenic mice were generated with the human CD2 (hCD2) gene linked to these deleted fragments. This allowed us to establish the minimum sequences necessary for the copy-dependent transgene expression. 2.1 kb or 1.5 kb of 3' flanking sequences linked to a hCD2 mini-gene is sufficient to allow T-cell specific, copy-dependent, integration-independent expression in transgenic mice. 1.1 kb of 3' sequences results in the gene being expressed in a T-cell specific manner, but copy-dependent, integration-independent expression was not observed in a small number of transgenic animals. 0.2 or 0.5 kb of 3' flanking sequences were insufficient to allow expression above the level previously found with a human CD2 gene which lacked 3' flanking sequences. We conclude that the Locus Control Region (LCR) effect is caused by 1.5 kb of flanking sequences immediately 3' to the polyadenylation signal of the gene.