Molecular cloning of KS, a novel rat gene expressed exclusively in the kidney.

BACKGROUND: We aimed to identify genes with kidney specific, developmentally regulated expression. Here we report the cDNA sequence and expression pattern of KS, a novel kidney-specific rat gene. METHODS: A partial cDNA was identified by differential display polymerase chain reaction (PCR) of a renal cell fraction enriched for proximal tubular and renin-expressing cells. Using the partial cDNA as a probe, a rat kidney cDNA library was screened. The full-length KS sequence was obtained by PCR amplification of cDNA ends. The expression pattern of KS was investigated by Northern blot. RNA was extracted from several organs of newborn and adult rats, as well as from the kidneys of rats with altered tubular function, that is, rats that had undergone unilateral nephrectomy, unilateral ureteral obstruction, neonatal losartan treatment, and the appropriate control animals. The expression of KS was also investigated in the kidneys of rats with spontaneous or renovascular hypertension. RESULTS: The KS cDNA (2426 bp) contained one open reading frame encoding a predicted 572 amino acid protein. The derived peptide sequence displayed approximately 70% similarity to the hypertension-related SA gene product and approximately 50% similarity to prokaryotic and eukaryotic acetyl-CoA synthases (EC 6. 2.1.1). KS was expressed in the kidney and not in any other organ assayed. KS RNA was not detected in fetal and newborn rat kidney but became apparent after one week of postnatal life. Gene expression was downregulated in rat models of altered tubular function. KS expression was decreased in spontaneously hypertensive rats but not in renovascular hypertension. CONCLUSION: KS, a novel rat gene, exhibits a unique tissue-specific expression exclusively in mature kidneys. The data suggest KS may encode an adenosine monophosphate binding enzyme.

Zis: a developmentally regulated gene expressed in juxtaglomerular cells.

Renal juxtaglomerular (JG) cells are specialized myoepithelioid cells located in the afferent arteriole at the entrance to the glomerulus. Their main function and distinctive feature is the synthesis and release of renin, the key hormone-enzyme of the renin-angiotensin system that regulates arterial blood pressure. Despite their relevance to health and disease, not much is known about factors that confer and/or maintain JG cell identity. To identify genes uniquely expressed in JG cells, we used a cell culture model and RNA differential display. JG cells cultured for 2 days express renin and renin mRNA, but after 10 days in culture they no longer contain or release renin and renin mRNA is reduced 700-fold. We report one cDNA differentially expressed in the 2-day JG cell culture that detects a 2.6-kb mRNA expressed at higher levels in newborn than adult kidney. Screening a 2-day culture JG cell cDNA library yielded clones representing differentially spliced transcripts. These cDNAs encode one unique protein (Zis) containing zinc fingers and domains characteristic of splicing factors and RNA binding proteins. Northern blot analysis confirmed Zis mRNA expression in differentiated JG cells, and identified an additional unique 1.5-kb transcript. The Zis transcripts are developmentally regulated in kidney and a number of other organs. The features of the Zis protein and its organ distribution suggest a possible role in regulation of transcription and/or splicing, both important steps for controlling developmentally expressed genes.

Design of four-helix bundle protein as a candidate for HIV vaccine.

To be efficient, a synthetic vaccine should contain different T and B cell epitopes of human immunodeficiency virus (HIV) antigens, and the B epitope regions in the vaccine and in the HIV should be conformationally similar. We have suggested previously the construction of vaccines in the form of a protein with a predetermined tertiary structure, namely a four-alpha-helix bundle. Antigenic determinants of cellular and humoral immunity are blocks for the vaccine design. From experimentally studied HIV-1 T and B cell epitopes, we constructed a sequence of a four-helix protein TBI (T and B cell epitopes containing immunogen). The gene of the protein was synthesized and the protein was produced in C600 Escherichia coli cells under recA promoter from Proteus mirabelis. CD spectroscopy of the protein demonstrated that 30% of amino acid residues adopt an alpha-helical conformation. Mice immunized with TBI have shown both humoral and cellular immune responses to HIV-1. The obtained data show that the design of TBI was successful. The synthesized gene structure makes possible further reconstruction and improvement of the protein vaccine structure.

[Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA]. / Identifikatsiia in vivo promotornoi aktivnosti v levom raione uchastka att DNK bakteriofaga lambda.

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.