The mouse homolog of PKD1: sequence analysis and alternative splicing.

We have cloned and sequenced the mouse transcript homologous to human polycystic kidney disease 1 (PKD1). The predicted protein is 79% identical to human PKD1 and shows the presence of most of the domains identified in the human sequence. Since the mouse homolog is transcribed from a unique gene and there are no transcribed, closely related copies as has been observed for human PKD1, we have been able to investigate alternative splicing of the transcript. At the junction of exons 12 and 13, several different splicing variants lead to a predicted protein that would be secreted. These forms are predominantly found in newborn brain, while in kidney the transcript homologous to the previously described human RNA predominates.

Distribution and developmentally regulated expression of murine polycystin.

PKD1, the gene that is mutated in approximately 85% of autosomal dominant polycystic kidney disease (ADPKD) cases in humans, has recently been identified (Eur. PKD Consortium. Cell 77: 881-894, 1994; also, erratum in Cell 78: 1994). The longest open-reading frame of PKD1 encodes polycystin, a novel approximately 460-kDa protein that contains a series of NH2-terminal adhesive domains (J. Hughes, C. J. Ward, B. Peral, R. Aspinwall, K. Clark, J. San Millan, V. Gamble, and P. C. Harris. Nat. Genet. 10: 151-160, 1995; and Int. PKD Consortium. Cell 81: 289-298, 1995) and several putative transmembrane segments. To extend studies of polycystin to an experimentally accessible animal, we have isolated a cDNA clone encoding the 3' end of Pkd1, the mouse homologue of PKD1, and raised a specific antibody to recombinant murine polycystin. This antibody was used to determine the subcellular localization and tissue distribution of the protein by Western analysis and immunocytochemistry. In the mouse, polycystin is an approximately 400-kDa molecule that is predominantly found in membrane fractions of tissue and cell extracts. It is expressed in many tissues including kidney, liver, pancreas, heart, intestine, lung, and brain. Renal expression, which is confined to tubular epithelia, is highest in late fetal and early neonatal life and drops 20-fold by the third postnatal week, maintaining this level into adulthood. Thus the temporal profile of polycystin expression coincides with kidney tubule differentiation and maturation.

The mouse homologue of the polycystic kidney disease gene (Pkd1) is a single-copy gene.

The mouse homologue of the polycystic kidney disease 1 gene (PKD1) was mapped to chromosome 17 using somatic cell hybrids, B x D recombinant inbred strains, and FISH. The gene is located within a previously defined conserved synteny group that includes the mouse homologue of tuberous sclerosis 2 (TSC2) and is linked to the alpha globin pseudogene Hba-ps4. Although the human genome contains multiple copies of genes related to PKD1, there is no evidence for more than one copy in the mouse genome. Like their human counterparts, the mouse Tsc2 and Pkd1 genes are arranged in a tail-to-tail orientation with a distance of only 63 bp between the polyadenylation signals of the two genes.

Structural motifs of the PKD1 protein.

The complete sequence of the polycystic kidney disease gene (PKD1) and its transcript have been described. The predicted protein is not a member of a previously described gene family, but contains several structural motifs that are present in proteins of known function. Most of these domains are present in the extracellular parts of proteins involved in interactions with other proteins and carbohydrates. The PKD1 gene product also contains potential transmembrane sequences. The molecule is likely to be involved in cell-cell or cell-matrix interactions, which is consistent with the different manifestations of polycystic kidney disease.

The TYE7 gene of Saccharomyces cerevisiae encodes a putative bHLH-LZ transcription factor required for Ty1-mediated gene expression.

In Saccharomyces cerevisiae, expression of a gene adjacent to the retrotransposon Ty1 is often mediated by Ty-internal sequences. We have identified novel mutants, tye7, which are affected in Ty1-mediated expression of ADH2 through a Ty1 sequence distal to the 5' long terminal repeat sequence. The TYE7 gene has been isolated and characterized. It encodes a 33 kDa protein whose N-terminal third is extremely rich in serine residues (28%). Within its C-terminal sequence, a remarkable similarity to Myc and Max proteins can be found. Thus, TYE7 is a potential member of the basic region/helix-loop-helix/leucine-zipper protein family. TYE7 function is not essential for growth. It may primarily function as a transcriptional activator in Ty1-mediated gene expression, as has been confirmed by the activation of reporter gene expression by a LexA-TYE7 hybrid protein. ADH2 activation by defined Ty1 derivatives revealed that TYE7 acts positively through the more distal Ty1 enhancer element (region D), and negatively in a region between A (the 5' proximal enhancer element) and D.

Isolation of the TYE2 gene reveals its identity to SWI3 encoding a general transcription factor in Saccharomyces cerevisiae.

The TYE2 gene was identified by recessive mutations which result in a significant reduction of Ty-mediated ADH2 expression. We cloned the TYE2 gene and analyzed its sequence. A large open reading frame of 825 codons was found encoding a rather hydrophilic, 93-kilodalton protein which contains a highly acidic region at its N-terminus. By sequence comparison we found that TYE2 is identical to gene SWI3 which has recently been shown to encode a nuclear protein which may function as a global transcription activation factor. The TYE2/SWI3 protein is necessary for the initiation of Ty1 transcription at its major initiation site in the delta element. Furthermore TYE2 function seems to be important for the expression of a variety of Ty-unrelated functions such as ADH1 expression, sporulation, growth on maltose, galactose, raffinose, and on non-fermentable carbon sources.

Characterization of trans-acting mutations affecting Ty and Ty-mediated transcription in Saccharomyces cerevisiae.

By recessive mutations, we have identified five genes, TYE1-TYE5, that are required for Ty-mediated expression of ADH2. These tye mutations not only suppress transcription of ADH2 when associated with a Ty element but are also defective in transcription of all Ty1 and Ty2 elements. Moreover, some of these mutations cause growth defects on non-fermentable carbon sources as well as sporulation defects. tye mutations also strongly suppress ADH2 expression when controlled by a polyA/T insertion mutation. Genetic analysis revealed that genes TYE3 and TYE4 are allelic to the previously identified genes SNF2 and SNF5 which code for transcription factors. These findings suggest that TYE gene products influence transcription of many genes rather than specifically Ty and Ty-mediated transcription. We have also found that null alleles of certain STE genes (ste7, ste11 and ste12), known to affect cell-type specific gene expression and expression of some Ty-adjacent genes, have a clear effect on Ty-controlled ADH2 expression depending on the carbon source. On the basis of ADH2 transcript levels in glucose-grown cells, all three ste alleles cause of five-fold reduction of ADH2 expression/transcription. In ethanol-grown cells, ste11 and ste12 mutations caused an almost complete loss of Ty-mediated ADH2 activation while ste7 has only a rather moderate effect. Surprisingly, ste11 and ste12 mutations lead to a significant increase in total Ty transcript levels. This would indicate that the STE12 protein, which is known to bind specifically to Ty1 sequences and thereby serve as an activator of a Ty-adjacent gene, can negatively modulate Ty transcription.(ABSTRACT TRUNCATED AT 250 WORDS)