Transgenic mice demonstrate novel promoter regions for tissue-specific expression of the urokinase receptor gene

The urokinase-type plasminogen activator receptor (u-PAR) contributes to cell migration and proteolysis in normal and cancerous tissues. Currently, there are no reports on the regulatory regions directing tissue-specific expression. Consequently, we undertook a study to identify novel promoter regions required for expression of this gene in transgenic mice bearing a LacZ reporter regulated by varying amounts (0.4, 1.5, and 8.5 kb) of upstream sequence. The 0.4-kb u-PAR upstream sequence directed weak and strong LacZ expression in the placenta and epididymis, respectively, both of which are tissues that express endogenous u-PAR. Conversely, transgene expression in the apical cells of the colon positive for endogenous u-PAR protein required 1.5 kb of upstream sequence for optimal expression. Furthermore, chromatin accessibility assays coupled with real-time polymerase chain reaction suggested a putative regulatory region spanning -1295/-1192 driving u-PAR expression in colonic cells. Interestingly, placental transgene expression was augmented with the 8.5-kb upstream fragment compared with the shorter 1.5-kb fragment indicating contributing element(s) between -1.5 and -8.5 kb. Thus, while 0.4 kb of upstream sequence directs u-PAR expression in the epididymis, sequences located between -0.4 and -1.5 kb and between -1.5 and -8.5 kb are required for optimal tissue-specific expression in the colon and the placenta, respectively.     

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans.

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.     

A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice

Erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located far upstream of the human beta-globin locus were inserted immediately upstream of a 4.1-kb fragment containing the human beta-globin gene. These constructs (HS beta) and a construct containing the beta-globin gene alone (beta) were microinjected into fertilized mouse eggs, and expression was analyzed in erythroid fetal liver and brain of day-16 embryos that developed. Only 7 of 23 animals that contained the beta gene alone expressed human beta-globin mRNA in erythroid tissue, and the average level of expression per gene copy was 0.3% of endogenous mouse beta-globin mRNA. In contrast, 50 of 51 transgenic mice that contained various HS beta constructs expressed the transgene specifically in erythroid tissue. The average level of expression per gene copy for constructs containing all five upstream HS sites was 109% of endogenous mouse beta-globin mRNA. Constructs that contained a single super-hypersensitive site (HS II beta) expressed 40% as much human beta-globin as mouse beta-globin mRNA per gene copy. These results demonstrate that the HS VI site, normally located downstream of the human beta-globin locus, is not required for high-level expression. Furthermore, the results demonstrate that high levels of human beta-globin gene expression can be obtained in transgenic mice even when a relatively small fragment of DNA (1.9 kb) containing erythroid-specific super-hypersensitive site II (HS II) is inserted upstream of the human beta-globin gene.     

Herpesvirus ICP18.5 and DNA-binding protein genes are conserved in equine herpesvirus-1

The genome of equine herpesvirus-1 (EHV-1) contained three open reading frames (ORFs) in a 3.9 kbpBamHI-SmaI fragment at 0.38–0.41 map units in the long unique region. The most 5′ ORF encoded the carboxy terminus of a protein with 45–55 percent amino acid homology to the DNA-binding proteins (ICP8-DBP) of four other alphaherpesviruses. The middle ORF translated to a polypeptide of 775 residues with 43–55% homology to the ICP18.5 proteins. The most 3′ ORF encoded the EHV-1 glycoprotein B (gB) gene. Three mRNAs of 4.3, 4.4–4.8, and 3.5–3.9 kb (corresponding to the three sequenced ORFs) were all transcribed from the same strand. The gene order of this group was conserved in all herpesviruses examined.     

Synergistic Effects of Alpha-Toxin and Perfringolysin O in Clostridium perfringens-Mediated Gas Gangrene

To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.     

Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.     

Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae.

The production of diphtheria toxin and siderophore by the Corynebacterium diphtheriae regulatory mutant C7(beta)hm723 is resistant to the inhibitory effects of iron, and the mutant strain is defective for function of the regulatory gene dtxR. A 2.8-kb HindIII fragment carrying the C7(beta)hm723 dtxR allele was cloned and characterized in Escherichia coli. The restriction endonuclease maps of the 2.8-kb HindIII fragment from C7(beta)hm723 and the corresponding fragment from wild-type C. diphtheriae C7 were identical. RNA dot blot analysis with total RNA isolated from wild-type C. diphtheriae C7 and C7(beta)hm723 indicated that the dtxR gene was transcribed at very low but equivalent levels in both strains and was not regulated by iron. beta-Galactosidase synthesis from a tox-lacZ translational fusion construct in E. coli in high-iron medium was not repressed by the C7(beta)hm723dtxR allele, but was strongly repressed by the wild-type dtxR gene. The 28- to 29-kDa polypeptide expressed from the mutant dtxR allele in E. coli had the same electrophoretic mobility as the wild-type dtxR gene product in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nucleotide sequence of the coding region and the 5' upstream region of the C7(beta)hm723 dtxR allele was determined and compared with the wild-type nucleotide sequence. The dtxR allele from C7(beta)hm723 contained a single-base change located 140 nucleotides from the 5' start of the gene, which resulted in replacement of arginine in the wild-type sequence by histidine in the mutant protein. These data demonstrate that C7(beta)hm723 expresses a mutant DtxR repressor protein that is severely defective in repressor activity.     

Glucocorticoids modulate human gonadotrophin releasing hormone upstream promoter activity in transfected human placental cells (JEG-3)

A human gonadotrophin releasing hormone (GnRH) upstream promoter/luciferase reporter gene construct (H2 construct) was generated by inserting a 1.7 kb XbaI/AflII fragment containing the human GnRH upstream promoter region only into a promoter-less luciferase reporter vector. When JEG-3 cells were transiently transfected with this construct and treated with cortisol or its synthetic analogue dexamethasone, a stimulatory effect on the upstream promoter activity was observed. This stimulation was dependent on the cotransfection of a glucocorticoid receptor (GR) cDNA expression vector due to the low level of GR in JEG-3 cells and could be completely abolished by RU486, a glucocorticoid antagonist. Moreover, the cortisol actions could be modulated to a different extent by oestradiol. Thus, since the human placenta contains GRs and the increase in cortisol metabolism near term is regulated by oestrogen, the current findings suggest that cortisol may be physiologically involved in the regulation of GnRH gene expression in the human placenta.      

A Segment of Human Vh Gene Locus is Duplicated

In humans, the kappa light chain variable region gene (Vk) locus evolved in part by duplications of four large segments. Similarly, some portions of the human heavy chain constant region gene locus are duplicated. Recently, we found that the Humhv3005 Vh gene is highly homologous to the reported 1.9III gene. Subsequently, restriction fragment length polymorphism study of the human Vh locus with a 1.6-kb EcoR1 fragment downstream of the hv3005 gene (termed hv3005/E1.6) suggested that the hv3005 and the 1.9III Vh loci might be generated by duplication from a common-ancestor Vh gene segment. To assess this possibility, we mapped the 15-kb region of the isolated hv3005 clone, beginning from 2 kb upstream, and sequenced the adjacent Vh4 gene (designated Humhv4005) located 10 kb downstream of the hv3005 gene. The result showed that hv4005 shared 99% homology with the 1.9II gene, located about 11 kb downstream of the 1.9III gene. Taken together, these data demonstrate that the hv3005-hv4005 region and the 1.9III-1.9II region arose by a duplication of a common ancestor Vh gene segment.     

Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli

The structural genes determining the edema disease principle were cloned from the total cellular DNA of Escherichia coli strain 412 (O139:K82) isolated from a case of porcine edema disease. An assay for cytotoxicity in Vero cells was used to detect the edema disease principle. A 7.5 kb EcoRI-SalI fragment specifying cytotoxin production was subcloned in pUC18. Sequences which specified production of cytotoxin were localized to a 0.9 kb region by transposon Tn5 mutagenesis. A 2.4 kb EcoRI-BglII fragment encompassing this region was subcloned into pUC18. Using nucleotide sequence analysis, two open reading frames separated by 12 bp were identified. They encoded proteins of 319 (A subunit) and 87 (B subunit) amino acids which both had N-terminal sequences typical of E. coli signal peptides. Comparison of these with the published sequence for the Shiga-like toxin II (SLT-II) showed 91% overall nucleotide sequence similarity. The nucleotide sequence similarity extended to 200 base pairs upstream of the putative A subunit translational start site suggesting a common regulatory mechanism. The deduced amino acid sequences of the processed A and B subunits had 94% and 84% similarity, respectively. These findings confirm the close genetic relationship between SLT-II and edema disease principle.     

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene

Adenosine deaminase (ADA) is expressed ubiquitously by diverse mammalian cells and tissues but at levels that vary according to tissue and species. In humans, the thymus exhibits levels of the enzyme up to 100-fold higher than most other tissues. Using transgenic mice, we identified human ADA gene regulatory domains. Up to 3.7 kb of 5'-flanking and first exon DNA from the human ADA gene failed to promote the expression of a chloramphenicol acetyl transferase (CAT) reporter gene in an efficient, reproducible, or tissue-appropriate manner in transgenic mice. However, when 12.8 kb of DNA from the first intron of the human ADA gene was placed 3' of CAT-coding and -processing sequences, transgenic mice reproducibly expressed CAT activity in most tissues, with profoundly high levels in the thymus. DNase I hypersensitivity studies demonstrated that among transgenic mouse tissues, human thymus, and a variety of human cell lines, a region of the intron 4-10 kb downstream of the first exon exhibited an array of hypersensitive sites that varied according to tissue and cell type. Deletion of this region from the gene construction eliminated high-level expression in transgenic mice. In transfection-transient expression assays, the 12.8-kb intron fragment exhibited enhancer activity in several cell types. A 1.3-kb fragment encompassing two of the hypersensitive sites exhibited some of these activities. The results of these studies suggest that the diverse pattern of human ADA gene expression is determined, in part, by a cluster of cis-regulatory elements contained within its large first intron.     

Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant.

Degenerate oligonucleotide primers were used in PCR to amplify a region of the recA homolog from Porphyromonas gingivalis W83. The resulting PCR fragment was used as a probe to identify a recombinant lambda DASH phage (L10) carrying the P. gingivalis recA homolog. The recA homolog was localized to a 2.1-kb BamHI fragment. The nucleotide sequence of this 2.1-kb fragment was determined, and a 1.02-kb open reading frame (341 amino acids) was detected. The predicted amino acid sequence was strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis. No SOS box, characteristic of LexA-regulated promoters, was found in the 5' upstream region of the P. gingivalis recA homolog. In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was insertionally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The recA-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results suggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivation of this gene did not alter the virulence of P. gingivalis in the mouse model.