摘要:

ABSTRACTBovine monocyte chemoattractant protein-1 (bovine MCP-1) cDNA has recently been characterized and shown to be highly expressed in bovine seminal vesicles secretory epithelium as well as in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear leukocytes (PMNLs). In an attempt to isolate the MCP-1 gene, we screened a bovine genomic cosmid library with a MCP-1-specific probe pH42. A positive clone, c11/1, was subjected to restriction analysis and fragments probed with pH42 by southern blotting. pH42-positive fragments were subcloned and sequenced. The sequence revealed three exon-like regions that coded for a protein displaying an identity of 51% with bovine MCP-1. Employing this sequence information from c11/1, the c11/1-specific cDNA was generated from poly(A)+RNA of bovine PMNLs by reverse transcription and a combination of polymerase chain reaction (PCR) methods. The assembled c11/1 cDNA comprised a 5′ UTR coding region as well as 3′ UTR for the gene product c11/1. Amino acid sequence comparison of the bovine c11/1 gene product with human monocyte chemotactic proteins yielded the highest sequence identity with hman MCP-2, and it is assumed that the c11/1 gene product represents the bovine MCP-2. The exon/intron structure of the bovine MCP-2 gene was found to be similar to the human MCP-1 gene. The bovine MCP-2 gene consists of three exons separated by two introns. In the 5′-flanking region of the 3.3-kb gene, a TATA box as well as an AP-1 sequence motif were identified. The bovine MCP-2 is specified by a single-copy gene. Contrary to MCP-1, MCP-2 is not expressed in bovine seminal vesicle tissue. In bovine PMNLs, expression of MCP-1 and MCP-2 is stimulated b

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.1

年代:1994

数据来源: MAL

摘要:

ABSTRACTrSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt −102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5′ upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additionalciselement (−645/−506). The positive and negativecis-elements together drive transcription of the chloramphenicol acetyltransterase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (−44/−29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (−44/−29) are competed by unlabeled −44/−29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides −40 to −38 and −34 to −32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-spec

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.9

年代:1994

数据来源: MAL

摘要:

ABSTRACTOur previous studies have shown that the hamster adrenal, like the human, contains high levels of preproenkephalin (PPenk) mRNA and enkephalin peptides, and may serve as a mammalian model for thein vivostudy of proenkephalin (Penk) gene expression, peptide biosynthesis, and release. To define further the factors that may regulate hamster Penk gene expression, the hamster Penk gene was isolated from a genomic library prepared from Syrian hamster liver. The hamster Penk gene contains four exons and three introns and encodes 268 amino acids including six copies of Met-enkephalin containing peptides and one copy of Leu-enkephalin. In the 5′ upstream region, there are TATA and GC boxes and multiple putative regulatory elements including the cAMP response element, AP-1, AP-2, AP-4, and the glucocorticoid response element (GRE). Possible GREs are also present in the introns. A comparison with the human and the rat Penk genes indicates that both the human and hamster Penk gene contain three introns, while the rat Penk gene has two introns. The intron missing from the rat Penk gene is short and separates the first and second exons of the hamster and human genes. In addition, the hamster and human genes share a region (100 bases) in the 5′ upstream sequence that is 98% homologous. It is of interest that Penk gene expression is high in the adrenal medulla of both human and hamster, but is much lower in the rat. These homologous regions and the extra intron may contain regulatory features responsible for a high level of expression in the human and hamster adrenal medu

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.25

年代:1994

数据来源: MAL

摘要:

ABSTRACTThe immunoglobulin J (joining) chain plays an important role in the assembly of polymeric immunoglobulins (dimeric IgA and pentameric IgM) and in the selective transport of these across epithelial cell layers. The primary structure of the bovine J chain has been determined by sequencing of three cDNAs. The cDNA has an open reading frame of 471 nucleotides encoding a putative protein of 157 amino acids. The 3′ untranslated region consists of 698 nucleotides and a poly(A) tail. The 5′ untranslated region and the promoter were isolated from a genomic clone. By comparison with the murine J chain gene, the 5′ untranslated region was predicted to be 37 bp, giving the bovine J chain cDNA a total length of 1,206 bp. This size was confirmed by Northern blot analysis of total RNA from colon and mammary gland. The amino acid sequence of the bovine J chain shows extensive homology with the J chain from human, mouse, rabbit, and bullfrog. Analysis of the J chain secondary structure showed a high propensity for forming β-sheets. An alignment of the predicted secondary structure of the J chain from bovine, human, mouse, rabbit, and bullfrog revealed a highly conserved "β-profile." The promoter sequence of the bovine J chain gene is presented and shown to contain a conserved interleukin-2 (IL-2)-responsive element previously characterized in the murine J cha

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.37

年代:1994

数据来源: MAL

摘要:

ABSTRACTRat microsomal epoxide hydrolase (mEH) is one of the detoxification enzymes and selectively expressed in liver. A 350-bp DNA fragment of the proximal promoter was found to contain information sufficient to express the mEH gene in hepatoma cells, however not in nonhepatoma cells. We identified twocis-acting elements, epoxide hydrolase proximal element 1 (EHP1) and 2 (EHP2), in this promoter region by using transient transfection assays. Each element is a new cell-type-specific transcriptional up-regulator. The cell-type-specific activity of EHP1 correlates to the limited cell distribution of its cognate transacting factor(s). In the case of EHP2, a similar or possibly the same cognate factor(s) binding to EHP2 was detected by DNase I footprinting and gel retardation assays in both hepatoma and nonhepatoma cells. However, EHP2 functions as an up-regulator only in hepatoma cells. Our finding adds repertoire to a battery ofcis-regulatory elements that are required for liver-specific transcription.

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.43

年代:1994

数据来源: MAL

摘要:

ABSTRACTThe intron of the yeastRP51Agene was cloned with precision using the polymerase chain reaction (PCR) amplification method, and then inserted into several different positions of the yeastURA3gene as well as thePGK–lacZfusion gene without introduction of additional exon sequences. Analysis of transcripts of these genes showed that an intron inserted near the transcription start site of the gene was spliced out efficiently, whereas the same intron sequences inserted 200 bp or further downstream of the start site were not, resulting in a reduced level of mRNA. These results explain why intron-containing genes in yeast usually have an intron near the 5′

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.51

年代:1994

数据来源: MAL

摘要:

ABSTRACTWe demonstrated earlier that post-exposure prophylaxis with 3′-azido-3′-deoxythymidine (AZT, zidovudine) or with AZT + interferon-α (IFN-α) prevented viremia and disease in BALB/c mice inoculated with Rauscher murine leukemia virus (RLV). After the 20-day treatment course, most animals were resistant to rechallenge with live virus. Adoptive transfer of T cells from such resistant but not from normal mice into naive recipients provided full protection against virus challenge. From these experiments, we concluded that post-exposure chemoprophylaxis restricted virus replication and allowed the animals to form protective, long-lasting cellular immune responses. Here, the role for cellular immunityduringantiviral chemoprophylaxis was tested by comparing treatment success in normal BALB/c mice and in their nude, athymic counterparts. Both were inoculated with equal doses of RLV (104plaque-forming units, pfu). Single-agent AZT or combination therapy with AZT + IFN-α, started before or after RLV inoculation, prevented viremia in all normal but not in most nude mice. A significant number of nude mice were completely protected by chemoprevention only when given a 10 times lower virus dose. When normal mice were injected with a 10 times higher virus dose (105pfu), complete protection by chemoprevention was lost. These results demonstrate that the success of chemoprevention depends critically on the virus inoculum. The differential success of chemoprevention in normal and T-cell-deficient mice implies that effective cellular immunity plays an important role in protecting virus-exposed animals against viremia and d

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.59

年代:1994

数据来源: MAL

摘要:

ABSTRACTRecently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5′-TCTGGTT-3′) in the HIV-1 LTR. Anin vivoCAT competition assay using −65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR r

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.67

年代:1994

数据来源: MAL

摘要:

ABSTRACTAmplification of DNA sequences using the polymerase chain reaction (PCR) requires as primers two oligonucleotides, which are carefully designed for length and G/C content. Such primers are generally between 18 and 30 bases long so that the primer sequences can amplify a unique sequence in the target genome; they should possess a minimal degree of secondary structure. We have tested the minimum length of G/C-rich and palindromic oligonucleotides to be used as primers in PCR. Oligonucleotides with sequences corresponding to the recognition sites of rare restriction enzymes were used on the DNA of vector constructs as model template DNA. Surprisingly, we found specific amplification with a low background over a wide range of temperatures for oligonucleotides as short as 7 nucleotides. This finding contradicts the previously reported empirical relationship between oligonucleotide length and ability to trigger amplification and points to the complex relationship between thermodynamic and kinetic criteria in relation to PCR. This technique should lead to new applications in the cloning and screening of complex genomes.

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.75

年代:1994

数据来源: MAL

摘要:

ABSTRACTA method is presented for efficient and large-scale isolation of plasmid DNAs from bacterial cells. Based on the cooperatively of heat and alkali actions, the method provides DNA preparations with high quality and yield (about 2 μg of DNA/ml culture), which are completely digestable by restriction enzymes and have a high transformation efficiency. Furthermore, the DNA preparations are extremely stable, and even through 4-year storage at −20°C, the electrophorogram and transformation efficiency remain as high as before. The factors affecting the stability of various DNA samples are discus

ISSN:1044-5498    

DOI:10.1089/dna.1994.13.83

年代:1994

数据来源: MAL