pE194, a 3.7-kilobase plasmid, confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. The previously identified cop and repF genes of pE194 have been further localized by molecular cloning and mutational analysis together with DNA sequencing. The CfoIB fragment of pE194 is capable of autonomous replication and contains both genes. Most of this region has been resequenced, and two errors reported in a previous study have been corrected. The corrected sequence indicates that the replication region contains a single large open reading frame, which we propose encodes the repF product. Northern blot (RNA blot) analysis of this region detected six transcripts, all transcribed in the same direction as one another and opposite to repF. A 350-base transcript is synthesized from the region containing cop. No in vivo transcript for the repF gene was detected, but a protein was observed in an in vitro transcription-translation system which appears to be its product. An ochre mutation was inserted in the putative repF open reading frame, and a nonsense fragment was detected in the in vitro system. When carried passively on a pUB110 replicon, this mutant product appears capable of inhibiting pE194 replicons in trans. The pE194 origin of replication has been localized to within 200 bases.
Abstract-Angiotensinogen is the glycoprotein precursor of 1 of the most potent vasoactive hormones, angiotensin II.Human angiotensinogen gene contains a C/A polymorphism at Ϫ20 located between the TATA box and transcriptional initiation site. We show here that when nucleoside A is present at Ϫ20, this sequence binds to the estrogen receptor.We also show that transcriptional activity of reporter constructs containing human angiotensinogen gene promoter with nucleoside A at Ϫ20 is increased on cotransfection of an expression vector containing human estrogen receptor-␣ coding sequence in human hepatoma cells (HepG2) followed by estrogen treatment. On the other hand, adenoviral major late transcription factor binds preferentially to this region of the promoter when nucleoside C is present at Ϫ20. We also show that reporter constructs containing human angiotensinogen gene promoter with nucleoside C at Ϫ20 have increased basal promoter activity on transient transfection in HepG2 cells as compared with reporter constructs with nucleoside A at Ϫ20. Our data suggest that C/A polymorphism at Ϫ20 may modulate the expression of human angiotensinogen gene in a sex-specific manner. (Hypertension. 1999;33:108-115.)Key Words: angiotensinogen Ⅲ genes Ⅲ polymorphism Ⅲ estrogen Ⅲ gene regulation Ⅲ regulation, hormonal Ⅲ major late transcription factor T he renin-angiotensin system plays an important role in the regulation of blood pressure, fluid balance, and electrolyte homeostasis. Angiotensin II, which is 1 of the most potent vasoactive hormones, is obtained from its precursor molecule, angiotensinogen, by the combined proteolytic action of renin and angiotensin-converting enzyme. Angiotensinogen is primarily synthesized in the liver, although recent studies have shown that its mRNA is also present in fat, brain, kidney, heart, and aorta of rats 1 and humans.2 Because plasma concentration of angiotensinogen is close to the Michaelis constant of the enzymatic reaction between renin and angiotensinogen, 3 a rise or fall in plasma angiotensinogen levels can lead to a parallel change in the formation of angiotensin II, and an increase in plasma angiotensin II may lead to hypertension. Previous studies have shown a highly significant correlation between plasma concentrations of angiotensinogen and blood pressure, 4 higher plasma angiotensinogen concentrations in hypertensive parents and their offspring, 5 and elevations of blood pressure in transgenic animals overexpressing the angiotensinogen gene. 6,7 Recent studies have suggested that the angiotensinogen gene locus is involved in human essential hypertension 8 and pregnancy-induced hypertension. 9Human angiotensinogen gene contains a C/A polymorphism at Ϫ20 located between the TATA box and transcriptional initiation site. 8 We show here that this region of the promoter binds to estrogen receptor-␣ when nucleoside A is present at Ϫ20. We also show that a reporter construct, pHAG1.2CAT (Ϫ20A), is transactivated by cotransfection of the mammalian expression vector pSG5 containing th...
Hypertension is a serious health problem in Western society, in particular for the African-American population. Although previous studies have suggested that the angiotensinogen (AGT) gene locus is involved in human essential hypertension, the molecular mechanisms involved in hypertension in African-Americans remain unknown. We show that an A/G polymorphism at ؊217 in the promoter of the AGT gene plays a significant role in hypertension in African-Americans. The frequency of the ؊217A allele was increased significantly in AfricanAmerican hypertensive subjects compared with normotensive controls. We also show that the nucleotide sequence of this region of the AGT gene promoter bound strongly to CAAT/enhancer-binding protein (C/EBP) family transcription factors when nucleoside A was present at ؊217. In addition, we show that reporter constructs containing the human AGT gene promoter with nucleoside A at ؊217 had increased basal transcriptional activity upon transient transfection in HepG2 cells compared with reporter constructs with nucleoside G at ؊217. Finally, we show that interleukin-6 treatment in the presence or absence of overexpressed C/EBP increased the promoter activities of reporter constructs containing nucleoside A at ؊217 compared with reporter constructs containing nucleoside G at ؊217. Because the AGT gene is expressed primarily in liver and adipose tissue, and C/EBP family transcription factors play an important role in gene expression in these tissues, we propose that increased transcriptional activity of the ؊217A allele of the human AGT gene is associated with hypertension in African-Americans.Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure (1-3). It is estimated that hypertension affects 50 million Americans with a prevalence rate of 25-30% in the adult Caucasian population, and the incidence of hypertension is even greater in the African-American population. Hypertension is a polygenic disease, and it has been estimated by segregation analysis and twin studies that ϳ45% of the interindividual differences in blood pressure can be accounted for by genetic differences. However, molecular mechanisms involved in the pathophysiology of human hypertension remain unknown. The renin-angiotensin system plays an important role in the regulation of blood pressure, and the octapeptide angiotensin II is one of the most active vasopressor agents (4, 5). Angiotensin II is obtained from its precursor molecule, angiotensinogen (AGT), 1 which is synthesized primarily in liver and adipose tissue and to a lesser extent in kidney, brain, heart, adrenal gland, and vascular walls (6, 7). AGT is first converted by renin to produce a decapeptide, angiotensin I, which is then converted to angiotensin II by the removal of a C-terminal dipeptide by angiotensin-converting enzyme. In experimental as well as clinical studies, administration of renin-angiotensin inhibitors is effective in reducing blood pressure and ending organ damage (8).Jeunemait...
A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolidelincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxy-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.
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