VACM-1 receptor is specifically expressed in rabbit vascular endothelium and renal collecting tubule.

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a novel arginine vasopressin (AVP) receptor that shares sequence homology with a cullin multigene family but not with the AVP receptors. To characterize the VACM-1 receptor, we examined its tissue-specific expression using Northern blot, RT-PCR, and immunostaining analyses. Northern blot hybridization identified a 6. 4-kb cRNA species that was expressed in the rabbit kidney medulla, brain, heart, and ovaries. In human tissue, VACM-1 mRNA is a larger (7.5 kb) cRNA found in the kidney, brain, heart, placenta, and skeletal muscle. VACM-1-specific RT-PCR products were detected in mRNA from rabbit kidney medulla, brain, heart, and mesenteric arteries. No expression of VACM-1 could be detected in rabbit aorta, gastrointestinal tract, or liver. Coimmunostaining with anti-VACM-1 antibodies (Ab) and a specific vascular endothelial cell marker, CD31 monoclonal Ab, localized VACM-1 expression to the vasculature in specific tissues. We identified the kidney cells expressing VACM-1 receptor by coimmunostaining with the following monoclonal Ab, which recognize epitopes in specific segments of the nephron: rct-30 Ab, reactive against the cortical and medullary collecting tubule (CT) cells; mr-omct Ab, reactive against the mitochondria-rich cells of the outer medullary CT; and an Ab specific against the loop of Henle segment. These studies indicated that the VACM-1 receptor is expressed only in the medullary CT. Kidney coimmunostaining with anti-VACM-1 and CD31 Ab identified VACM-1-receptor expression in glomeruli and medullary vascular bundles. These results demonstrate that the novel VACM-1 receptor, expressed in many organs, is localized to the endothelial cells. In the kidney, it is also expressed in the medullary CT cells. Thus VACM-1 may be involved in the regulation of endothelial permeability and water transport in the CT.

Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization.

We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5' region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli, (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5' end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3' end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions - fibrillar centers, dense fibrillar components, and granular components - for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.

Infectivity and complete nucleotide sequence of the cloned genomic components of a bipartite squash leaf curl geminivirus with a broad host range phenotype.

Through cloning and molecular analysis we have identified two highly homologous bipartite geminiviruses as causing squash leaf curl disease. Mechanical and Agrobacterium-mediated inoculation of plants with cloned viral DNA components identified the two genomic components of SqLCV-E, a squash leaf curl virus with an unexpectedly broad host range for a whitefly-transmitted geminivirus. Nucleotide sequence analysis of the genome of this virus showed it to have the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Sequence comparison with the genomic components of tomato golden mosaic virus and bean golden mosaic virus revealed a close evolutionary relationship with these two bipartite geminiviruses, with which SqLCV-E shares common hosts. These studies provide clear molecular evidence for the assignment of SqLCV to the subfamily of bipartite geminiviruses.

Autonomously replicating sequences in young and senescent mitochondrial DNA from Podospora anserina.

Senescence in the filamentous fungus Podospora anserina is characterized by the accumulation of multimeric circular mitochondrial DNA molecules, known as senDNAs. These tandemly repeated DNA sequences, which originate from broadly dispersed regions of the young mitochondrial genome, behave as independently replicating molecules. In this study, the yeast transformation system was used to assay senDNAs and their young mtDNA counterparts for the presence of autonomously replicating sequences. P. anserina mtDNA fragments were cloned into the yeast vector YIp5 and the hybrid YPM plasmids were used to transform yeast. All of the senDNAs and their homologous young mtDNAs promoted high frequency transformation and extrachromosomal maintenance of YPM plasmids. The putative origin of replication for the P. anserina mitochondrial genome was also cloned into YIp5 and shown to confer autonomously replicating properties.