Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.     

Structure of the human immune interferon gene

Sequence determination of cloned cDNAs and genes of the three classes of interferon (IFN-alpha, -beta and -gamma) has revealed more than a dozen members of the human IFN-alpha gene family and a single gene for IFN-beta. These genes are found on chromosome 9 and contain no introns. We recently reported that the 146-amino acid sequence of mature IFN-gamma deduced from the nucleotide sequence of a cloned cDNA was quite unrelated to those of the other IFNs, and that the gene for IFN-gamma contains at least one intron. We now describe the isolation, characterization and DNA sequence of the human IFN-gamma gene. It contains three introns, a repetitive DNA element, and is not highly polymorphic. All our evidence to date and the present data suggest that this is the only gene for IFN-gamma and that the resolution of IFN-gamma into two components is probably the result of post-translational processing of the protein.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Different antiviral spectra of human macrophage interferon activities

The antiviral activity of alpha-interferon (IFN-alpha) produced by human leukocytes in response to viral infection has been considered to be independent of the virus which induced its production. Recently, however, IFN-alpha has been found to include at least eight different subtypes, as indicated by measurement of antigenic variability, DNA hybridization and amino acid sequencing. We considered the possibility that interferon heterogeneity may play a part in enhancing host antiviral defence and now present data suggesting that purified human macrophages, when exposed to different viruses, produce interferons having differing spectra of antiviral activity. These findings may provide a functional correlation for IFN-alpha heterogeneity.     

Is sequence conservation in interferons due to selection for functional proteins?

The human alpha-interferon (IFN-alpha) gene family consists of at least 14 potentially functional non-allelic members; the amino acid sequences they encode differ from each other by up to approximately 20% of their residues. Human IFN-beta, which is encoded by a single gene, is distantly related to the IFN-alpha family; it differs in 67% of its residues from IFN-alpha 2. There is considerable evidence that IFN-alpha and -beta compete for the same receptors on their target cells. Comparison of 14 non-allelic human IFN-alpha sequences and the IFN-beta sequence has revealed that 37 of 166 residues are completely conserved and that several of these are arranged in clusters, for example at positions 29-33, 47-50 and 136-150. It is commonly held that evolutionary conservation of amino acids indicates that the residues in question are essential for function. To test this hypothesis in the case of IFNs, we have introduced single site-directed point mutations into the strictly conserved codons 48 and 49 of the IFN-alpha 2 gene which form part of the longest uninterrupted cluster (position 47-50). We report here that the mutant proteins, containing Tyr, Ser and Cys instead of Phe48, or His instead of Gln49, have biological activities indistinguishable from those of wild-type IFN-alpha. In addition, when Glu62, a residue conserved in all known alpha and beta IFNs of man, mouse and cattle, was replaced by Lys, antiviral activity remained unchanged.     

Interactions between the nuclear matrix proteins and the 5'-flanking cis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMPk) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE Ⅱ , - 446- - 419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay. Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMPk in K562, cells may be composed of two polypeptides ( ～ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA ( - 392- - 177 bp) in the 5'-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.     

A point mutation in the A gamma-globin gene promoter in Greek hereditary persistence of fetal haemoglobin

Hereditary persistance of fetal haemoglobin (HPFH) is a benign condition characterized by the production in adulthood of more than 1% fetal haemoglobin (HbF, alpha 2 gamma 2) in the absence of erythropoietic stress. Several genetic types have been discerned based on the level of HbF produced, the relative contributions of the duplicated fetal (G gamma and A gamma) globin genes, and the presence or absence of deletions involving the beta and delta genes in cis to the mutation. Greek HPFH is a non-deletion variety in which heterozygotes produce 10-20% HbF, predominantly due to overproduction of the A gamma chain. We have cloned a 40-kilobase (kb) region of the beta-globin cluster from a Greek HPFH allele and report here that a point mutation (G----A) occurs 117 base pairs (bp) 5' to the cap site of the A gamma-globin gene, just upstream of the distal CCAAT sequence. The corresponding region of the G gamma-globin gene is normal. We discuss the implications of this finding for the developmental regulation of globin gene expression.     

The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha

The 12 interferon (IFN)-related sequences detected in a human gene bank fall into not less than eight distinct classes, indicating that there are at least eight IFN-related genes. Most, if not all, of these direct the synthesis of an IFN in Escherichia coli. The sequence of one chromosomal gene and its flanking regions was identical to that deduced for the cDNA corresponding to IFN-alpha l mRNA. No evidence was found for the existence of an intron, in either the coding or the non-coding segments of the gene.     

Discrete cis-active genomic sequences dictate the pituitary cell type-specific expression of rat prolactin and growth hormone genes

The anterior pituitary gland, which is derived from a common primordium originating in Rathke's pouch, contains phenotypically distinct cell types, each of which express discrete trophic hormones: adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), prolactin, growth hormone, and follicle stimulating hormone (FSH)/luteinizing hormone (LH). The structurally related prolactin and growth hormone genes, which are evolutionarily derived from a single primordial gene, are expressed in discrete cell types--lactotrophs and somatotrophs, respectively--with their expression virtually limited to the pituitary gland. The pituitary hormones exhibit a temporal pattern of developmental expression with rat growth hormone and prolactin characteristically being the last hormones expressed. The reported co-expression of these two structurally related neuroendocrine genes within single cells prior to the appearance of mature lactotrophs, in a subpopulation of mature anterior pituitary cells, and in many pituitary adenomas raises the possibility that the prolactin and growth hormone genes are developmentally controlled by a common factor(s). We now report the identification and characterization of nucleotide sequences in the 5'-flanking regions of the rat prolactin and growth hormone genes, respectively, which act in a position- and orientation-independent fashion to transfer cell-specific expression to heterologous genes. At least one putative trans-acting factor required for the growth hormone genomic sequence to exert its effects is apparently different from those modulating the corresponding enhancer element(s) of the prolactin gene because a pituitary 'lactotroph' cell line producing prolactin but not growth hormone selectively fails to express fusion genes containing the growth hormone enhancer sequence.     

Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.     

水稻矮化突变体G蛋白α亚基基因的结构和表达

利用γ-Co60辐射诱发水稻特光矮-2(Oryza sativa L.cv.TGA-2)产生变异,获得一种稳定遗传的新型水稻矮化突变体dwarf69.dwarf69和TGA-2及其杂交后代F1、F2、F3成熟期的株高数据表明矮化表型受一对隐性基因控制.进一步研究发现,虽然dwarf69和TGA-2的G蛋白α亚基基因(Rice G protein alpha-subunit,RGA)编码区核苷酸序列只有一个核苷酸的差异,但RGA在野生型TGA-2中的表达量明显高于在突变体dwarf69中的表达量.对矮化突变体dwarf69和野生型TGA-2的RGA基因5＇上游区的序列分析表明,dwarf69 RGA 5＇上游区比TGA-2RGA5＇上游区多出1076bp.首次报道水稻矮化突变体中的RGA5＇上游区序列与其野生种的RGA5＇上游区序列存在显著的差异.