Upstream sequences within the terminal hairpin positively regulate the P6 promoter of B19 parvovirus

For the B19 parvovirus P6 promoter, a 96-nt minimal truncation mutant retained activity in transient reporter gene assays. Deletion of sequences further upstream from this minimal promoter markedly diminished reporter activity in certain cell lines. This upstream region lies within the terminal hairpin from -249 to -157 and contains a 14-nt sequence that is protected by DNase I footprinting. The exact sequence is directly repeated further within the hairpin, suggesting a regulatory role. The hairpin termini of parvoviruses were known to serve as origins of replication and to catalyze virion packaging. We now suggest that, in addition to these functions, they exert cis-acting effects on B19 P6-promoted gene expression.     

Phloem specific promoter from a satellite associated with a DNA virus

DNAbeta is a satellite molecule associated with some monopartite begomoviruses and encodes a single gene (betaC1), which is highly conserved in position and size among DNAbeta molecules. A 955 nt fragment of Tomato yellow leaf curl China virus (TYLCCNV) DNAbeta, upstream of the translation start site of betaC1 gene was tested for its promoter activity with gus as a reporter gene. Analysis of beta-glucuronidase (GUS) activity following transient expression assays indicated that the 955 nt fragment had promoter activity and that 3'-deletions of 399 or 173 nt of the fragment resulted in complete loss of its promoter activity. The 5'-deletions of 782 or 556 nt of the fragment, however, did not affect its activity. Histochemical staining revealed that this fragment can be used to express gus gene specifically in phloem tissue of stably transformed tobacco plants. Further studies have indicated that a 173 nt segment from 3'-end of the 955 nt fragment was responsible for basic promoter activity and phloem-specific expression.     

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.     

Dugbe nairovirus M RNA: Nucleotide sequence and coding strategy

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.     

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.     

Primary structure and gene structure of staphylokinase

The nucleotide sequence was determined of the 2.9 kb HindIII restriction fragment, obtained from the genomic DNA of a selected Staphylococcus aureus strain (strain no. 23) which directs high level expression of staphylokinase (STA) in E. coli JM83 cells transformed with the recombinant plasmid pUCSTAHH, consisting of pUC19 containing this 2.9 kb insert. The fragment contained an open reading frame of 489 base pairs (base pairs 996–1484) encoding 163 amino acids, with amino acids 28, 34 and 38 corresponding to the NH₂-terminal residue of the three variants (STA-M, STA-Δ6 and STA-Δ10) of recombinant STA (STAR) recovered from culture broth conditioned by transformed E. coli (Collen et al, Fibrinolysis, 1992; 6: 203–213). This coding sequence is preceded upstream by canonical Shine-Dalgarno, -10 and -35 prokaryotic promoter sequences and in addition by an open reading frame spanning nucleotides 50–802 which encodes an unknown protein of 251 amino acids. The sequence encoding STA is very similar to that previously cloned from bacteriophages Sø-c and 42D. Recombinant plasmids with a l.7kb AccI-EcoRI fragment insert (base pairs 683–2168) containing only the sequence encoding STA, directed excretion of STA activity to an extent comparable to that obtained with the recombinant plasmid containing the 2.9 kb HindIII restriction fragment insert.