摘要:

Serum amyloid A (SAA) is highly induced during many inflammatory episodes. The induction mechanism in response to turpentine and lipopolysaccharide (LPS), two major inducers of this gene, was investigated. Here we present evidence that although both agents triggered expression, SAA mRNA synthesized in the turpentine-injected rabbit liver is many-fold higher compared to that found in LPS-injected rabbit liver. We demonstrate that differential level of activation of C/EBP and NF-κB that interact with the proximal promoter of SAA gene is responsible for the differential expression. A very high level of C/EBP induction with little or no activation of NF-κB factors was noted when turpentine was used as the inducer. LPS, on the other hand, activated NF-κB and C/EBP, which were detected only at the early phase of induction process. These results indicate that different pathways might be activated for the regulation of hepatic expression of SAA by different inflammatory agents. One of the pathways, triggered by LPS, requires participation of both NF-κB and C/EBP. A second pathway, triggered by turpentine, involves only C/EBP family of transcription fact

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.1

年代:1997

数据来源: MAL

摘要:

Plastins (or fimbrins) are a family of actin-binding proteins that are conserved from yeast to humans. In mammals, three tissue-specific plastin isoforms have been identified. The L isoform (L-plastin) is normally expressed only in leukocytes but is also found in>90% of neoplastic nonleukocyte human cells. Because L-plastin expression in tissue-specifically regulated in both humans and rodents, it is likely that similar mechanisms regulate L-plastin gene expression in human and rodent cells and that they could be identified by comparing the function and nucleotide sequences of the human and murine L-plastin gene promoters. Previously, we reported the isolation and characterization of the human L-plastin gene promoter. In this study, we isolated a murine L-plastin 5′ end cDNA and used it as a probe to isolate several murine genomic clones. A representative clone contained 7 kb of the flanking region, 0.1 kb of the first exon, and 9.9 kb of the first intron. A continuous 1,354-bp sequence was identified around the first exon. Five transcription initiation sites were found 40 to 73 bp downstream from a perfect TATA box. Alignment of the sequence with its human counterpart revealed ~60% homology in a 1-kb region spanning the first exon and the flanking region. The TATA box, one ER binding site, and two ETS binding sites were completely conserved. An Spl binding sequence in the human promoter was partially conserved in the murine promoter but could still bind to Spl. A second ER binding sequence, lying 5′ adjacent to the TATA box in the human promoter, was conserved only at the 3′ half-site in the murine promoter; the 5′ half-site was changed into a potential AP1 binding site. This AP1/ER hybrid sequence was incapable of binding to ER. However, both human and murine promoters were found to function equally well in either human or murine leu

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.9

年代:1997

数据来源: MAL

摘要:

Mouse liver cell lines that bear a stably integrated lactose operon repressor (lacl) gene and a Ha-rasgene linked to a lactose operator-containing SV40 early promoter were generated. When grown in medium containing more than 0.1 mMisopropyl β-D-thiogalactoside (IPTG), the Ha-rasgene was induced up to 20-fold. Maximum induction of Ha-rasgene expression occurred after 12 h of exposure. The tumorigenicity of these cell lines in syngeneic mice was enhanced when the mice were maintained on drinking water containing 12.5 mMIPTG. Ha-rasgene expression in tumors was strongly induced in the presence of IPTGin vivo. Induction of Ha-rasgene expression in mice was consistently observed after 48 hr of exposure to drinking water containing IPTG. This system provides an approach for studying the function of oncogenein vivoas well as other genes of interest

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.17

年代:1997

数据来源: MAL

摘要:

Prosaposin is a multifunctional protein that, when secreted, functions as a neurotrophic agent and, when retained in the lysosomes, is processed to essential glycosphingolipid hydrolase activator proteins. The prosaposin locus is temporarily and spatially regulated at the transcriptional and post-translational levels. The prosaposin gene has been partially characterized, but the 5′ region has not. RACE, S1nuclease protection, and sequence analysis were used to characterize the first intron and first exon as well as the 5′-flanking regions from murine P1 clones. The first intron is ~15 kb in length and the complete gene is ~25 kb. The transcriptional initiation sites are located 87 and 94 bp 5′ to the ATG in exon 1. Using luciferase as a reporter gene and transfection into NS20Y, NIH-3T3, or SF-7 Sertoli cell cultures, deletion constructs from the 5′ putative promoter region were shown to contain positive and negative regulatory elements within 2,400 bp 5′ to the transcription start site. A negative regulatory element is located between 742 and 310 bp 5′ to the transcription start site. These studies provide insight into the regulation of this unique "lysos

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.23

年代:1997

数据来源: MAL

摘要:

Members of the low-density lipoprotein receptor (LDLR) supergene family interact with a large number of diverse ligands. One of the relevant receptors is the recently characterized LDLR relative with eight ligand-binding repeats, termed LR8, which exists in two splice variant forms. The gonads, relying on receptor-mediated lipoprotein supply for steroidogenesis, and on interplay of germ cells with somatic cells, provide a particularly attractive setting to study details of the expression of LR8. Here we show by polymerase chain reactions and Northern analysis, as well as byin situhybridization, that the longer of the two splice variants (LR8+), containing an additional region defining anO-linked sugar domain, is produced in the somatic cells of chicken testis, whereas the shorter form lacking this domain (LR8-) is expressed in the male germ cells. Interestingly, as shown by transcript analysis and at the functional level by ligand blotting, LR8- expression in the spermatoids increases with germ cell maturation, but is absent from ejaculated sperm. This constitutes a scenario reminiscent of the situation in growing vitellogenic oocytes, which express very high levels of LR8-, but lack the receptor following ovulation. Thus, the cell-specific expression of different LR8 splice variants may relate to the requirements of extensive communication and cooperation between germ cells and somatic cells in the gonads.

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.35

年代:1997

数据来源: MAL

摘要:

Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, bothin vitroandin vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within theSlpenhancer context increased Oct-1 bindingin vitro, but greatly reduced hormonal inductionin vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C′Δ9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C′Δ2). This underscores that, despite extensive sequence identity of C′Δ9 and C′Δ2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.45

年代:1997

数据来源: MAL

摘要:

The diazepam binding inhibitor [DBI, also known as acyl-CoA-binding protein, (ACBP), or endozepine] is a 10-kD protein that has been suggested to be involved in the regulation of several biological processes such as acyl-CoA metabolism, steroidogenesis, insulin secretion, and γ-aminobutyric acid type A (GABAA)/benzodiazepine receptor modulation. DBI has been cloned from vertebrates, insects, plants, and yeasts. In mammals, DBI is expressed in almost all the tissues studied. Nevertheless, DBI expression is restricted to specific cell types. Here we have studied DBI gene expression in the germ-line cells of rat testis. The DBI gene was intensively transcribed in postmeiotic round spermatids from stages VI to VIII of the seminiferous epithelial cycle. A prominent, spermatid-specific upstream transcription initiation site was identified in addition to the multiple common transcriptional initiation sites found in the somatic tissues. However, no DBI protein was detected in round spermatids, suggesting that the DBI transcripts were translationally arrested. The DBI protein was detected in the late spermatogenic stages starting from elongating spermatids from step 18 (stage VI) onward. The DBI protein was also detected in mature spermatozoa and in ejaculated human sperms. The majority of DBI was located at the middle piece of the spermatozoons tail enriched with mitochondria. On the basis of this observation and the well-established role of DBI in acyl-CoA metabolism, we propose that DBI expression in spermatozoa reflects the usage of fatty acids as a primary energy source by spermatozoa. The biological function of DBI in spermatozoa could thus be related to the motility function of sperm

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.59

年代:1997

数据来源: MAL

摘要:

Human uteroglobin (hUG) or Clara cell 10-kD protein (cc10 kDa) is a steroid-dependent, immunomodulatory, cytokine-like protein. It is secreted by mucosal epithelial cells of all vertebrates studied. The cDNA encoding hUG and the 5′ promoter region of the gene have been characterized previously. Here, we report that the structure of the entire hUG gene is virtually identical to those of rabbit, rat, and mouse. It is localized on human chromosome 11ql2.3-13.1, a region in which several important candidate disease genes have been mapped by linkage analyses. Our data indicate that candidate genes for atopic (allergic) asthma and Best's vitelliform macular dystrophy are in closest proximity to the hUG gene. To determine whether hUG gene mutation may be involved in the pathogenesis of these diseases, we studied two isolated groups of patients, each afflicted with either atopy or Best's disease, respectively. We detected a single base-pair change in the hUG gene in Best's disease patients and normal controls but no such change was detected in atopy patients. This alteration in hUG gene-sequence in Best disease family appears to be a polymorphism. Although the results of our investigation did not uncover mutations in hUG gene that could be causally related to the pathogenesis of either of these diseases, its conservation throughout vertebrate phyla implies that this gene is of physiological importance. Moreover, the close proximity of this gene to several candidate disease genes makes it an important chromosomal marker in cloning and characterization of those gene

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.73

年代:1997

数据来源: MAL

摘要:

Genomic DNA sequences encoding the murine Janus family tyrosine kinase Jak3 were isolated to determine the intron-exon structure of the gene and to investigate the phylogeny of Jak-family kinases. The murineJak3gene comprises approximately 15 kbp of genomic DNA and consists of 23 exons. The organization of sequences encoding the pseudo-kinase domain of Jak3 is similar to the intron-exon structure encoding catalytic domains of Src-family tyrosine kinases, whereas the pattern of introns-exons encoding the Jak3 kinase domain shows no structural similarity to that of other tyrosine kinase genes. Genomic analysis further indicates that alternative splicing gives rise to different forms of the murineJak3mRNA encoding different isoforms of the Jak3 protein. Analysis ofJak3intron-exon structure also suggests that a mutation in the humanJAK3gene responsible for a severe combined immune deficiency (SCID) phenotype results from aberrant splicing of theJAK3transcript. Finally, potential regulatory sequences in the upstream region of the murineJak3gene were analyzed and are discussed in relation to the known expression pattern of Jak3.

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.85

年代:1997

数据来源: MAL

摘要:

The chicken growth hormone-releasing hormone (GRF) gene was isolated, sequenced, and characterized. In addition, three different mRNAs were isolated from juvenile and adult brain. The first cDNA encoded for a GRF1-46, the second cDNA encoded for a GRF1-43due to a sliding intron boundary, and the third skipped exon four and encoded only GRF33-46. We also determined that juvenile chicken mRNA encoding GRF is expressed in the brain and gonads, but not in the pituitary, heart, liver, kidney, crop, small intestine, large intestine, eye, and muscle. This gene is also interesting in terms of evolution because another neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is encoded within the same gene (grf/pacap) in chicken, but on a separate gene (pacap) in mammals. We showed previously that these two neuropeptides were encoded in the same cDNA in fish, but the present evidence in chicken suggests a gene duplication in stem mammals.

ISSN:1044-5498    

DOI:10.1089/dna.1997.16.95

年代:1997

数据来源: MAL