©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ke 6 Gene
SEQUENCE AND ORGANIZATION AND ABERRANT REGULATION IN MURINE POLYCYSTIC KIDNEY DISEASE (*)

(Received for publication, July 20, 1995)

Michele M. Maxwell (1)(§) Jacqueline Nearing (1) Nazneen Aziz (1) (2)(¶)

From the  (1)Nephrology Division, Department of Medicine, Children's Hospital and the (2)Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ke 6 gene is a newly identified gene located in the major histocompatibility complex and is a candidate steroid dehydrogenase gene because of structural homology and regulatory similarities with mammalian steroid dehydrogenases. We report here the complete nucleotide sequence and intron-exon organization of the Ke 6 gene and cloning of the alternatively spliced Ke 6b transcript. We find that Ke 6 gene expression is down-regulated in pcy mice which is a murine model of polycystic kidney disease (PKD). Thus far, Ke 6 gene expression is down-regulated in all murine models of PKD we have examined. Abnormal steroid metabolism as a possible cause of PKD is discussed.


INTRODUCTION

Polycystic kidney disease (PKD) (^1)is a major inherited cause of renal failure in humans. The occurrence of the autosomal dominant form of the disease is very high with a frequency of 1 in 1,000(1) . Recently, the ADPKD1 gene was isolated and sequenced, but the nature of the protein it encodes is as yet unknown(2) . However, it is clear that polycystic kidney disease is a multigenetic disease, since numerous inherited diseases occur in both humans and mice which give rise to renal cysts caused by different genes. In rodents, several genes have been identified e.g. cpk, pcy, jck, cy, bpk, jcpk, TgN737, and Bcl-2 which give rise to polycystic kidneys(3) . In humans there is a second gene (ADPKD2) responsible for the dominant form of the disease, located on chromosome 4(4, 5) . Autosomal recessive polycystic kidney disease is a rarer, although more aggressive, form of PKD in man, where the mutation has been mapped to chromosome 6(6) . There are several other lesser known heritable diseases, such as Meckle's syndrome, Jeune's Syndrome, and Von Hipple Lindau Syndrome, where cysts occur in the kidney by what may be yet other cyst causative genes. Renal cysts can also occur in long term dialysis patients,. and this form of the disease has been termed acquired PKD. To date the biochemical and molecular basis of renal cysts formation is virtually unknown.

We reported the identification of a new mammalian gene, Ke 6, whose expression is specifically repressed in two different murine models of PKD: the cpk and jck mouse(7) . The Ke 6 gene is encoded within the major histocompatibility complex and is a member of the superfamily of short-chain alcohol dehydrogenases. The normal expression pattern of the Ke 6 gene is very high in kidney and liver (7) , the two organs that are most extensively affected in polycystic kidney disease. We have postulated that the Ke 6 gene may encode steroid dehydrogenase activity, since it has substantial homology to specific functional domains of bacterial and mammalian steroid dehydrogenases(8) . In addition, the pattern of down-regulation of the Ke 6 gene is remarkably similar to the 11beta-hydroxysteroid dehydrogenase gene in liver and kidneys of jck and cpk mice(8) . Steroid dehydrogenases are enzymes that metabolize biologically active forms of hormone to their inactive keto derivatives. The possibility that Ke 6 is a steroid dehydrogenase is therefore very exciting, especially since a role of steroids in cystic malformation of nephrons is well established. We have postulated that the primary genetic defect in the cpk and jck homozygous mice leads, either directly or indirectly, to the repression of the Ke 6 gene(7, 8) . The lowered activity of Ke 6 gene expression could lead to a deficiency of a steroid metabolic enzyme and consequently result in elevating the intrarenal steroid levels. The increased concentration of biologically active steroids is most likely responsible for initiating events that ultimately result in polycystic kidney disease(8) . Steroids administered to neonate rodents are known to induce cystic maldevelopment of nephrons in vivo(9, 10, 11, 12, 13, 14) , and in vitro, in organ cultures of metanephric kidney(15) . Elevated concentration of steroids could be detrimental to developing kidney during embryogenesis and during postnatal differentiation. Even though in numerous studies steroids have been implicated in forming renal cysts, the specific molecular mechanism of steroids' action in cyst formation is unknown.

In order to understand the function of the Ke 6 gene and the mechanism of its normal and aberrant regulation in cpk and jck mice, it is essential to characterize the transcriptional unit of Ke 6 gene in greater detail. Characterization of the Ke 6 gene promoter sequence offers the possibility of investigating the interaction of transcription factors and cis-regulatory elements responsible for its high level of expression in kidney and liver and its aberrant regulation in PKD. We report here the cloning and sequencing of an alternatively spliced mRNA Ke 6b and complete sequence determination of the Ke 6 gene and its intron/exon organization. We used RNase protection assay and primer extension analysis to determine the transcriptional initiation site of the gene. Putative regulatory elements in the promoter region were identified by searching the transcriptional regulatory sequence data base at National Center for Biotechnology Information.

In addition, we find that Ke 6 gene expression is down-regulated in mice carrying the pcy mutation. The pcy mouse is another murine form of PKD which manifests in homozygous juveniles. Ke 6 is down-regulated in all murine models of PKD, congenital and juvenile, that we have examined so far. This indicates that Ke 6 most likely represents a critical steroid metabolic enzyme in normal kidney function whose reduction can be deleterious not only to post-natal development of nephrons but also for the maintenance of healthy renal tubules.


MATERIALS AND METHODS

Genomic DNA Isolation and Southern Blot Analysis

Genomic DNA was isolated from C57BL/6 mouse liver as described previously(16) . 20 µg of DNA was restricted and run on a 0.8% agarose gel. DNA was blotted in 10 times SSC onto a nylon membrane (Magna Graph, MSI) and UV-cross-linked using a Stratalinker (Stratagene). Prehybridization and hybridization were in Church's buffer (0.5 M sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, 1 mM EDTA, and 100 µg/ml salmon sperm DNA) at 65 °C. A alpha-P-labeled insert from the Ke 6a cDNA clone p13.1.1 was added to hybridization buffer at 10^6 cpm/ml. The membrane was washed in 0.2 times SSC, 0.1% SDS at 50 °C.

Northern Blot Analysis

Poly(A) RNA was extracted from kidneys and liver of a 4-5-week-old C57BL/6 +/+ male, a 12-week-old DBA pcy/pcy female, a 8-week-old C57BL/6 jck/jck male (kidney), a 25-week-old C57BL/6 jck/jck male (liver), and a 19-day-old C57BL/6 cpk/cpk female mice as described previously(7) . Poly(A) RNA was separated on a 1% agarose/formaldehyde gel followed by blotting onto a nylon membrane (Magnagraph, MSI). Hybridization was carried out as described for genomic blot. DNA probes were labeled by the random-primed labeling method(17) , cleaned by spin column, and added to the hybridization fluid at 10^6 cpm/ml. Membranes were washed at 50 °C in 0.1 times SSC, 0.1% SDS.

Isolation, Subcloning, and Sequencing of Ke 6 Genomic DNA

A recombinant phage MS-I containing the Ke4/Ke6 loci was isolated by screening a D3 mouse ES cell line genomic library using the insert from Ke 4 cDNA. A 7.7-kb KpnI fragment from phage MS-I encompassing the entire Ke 6 gene and the 3` end of the Ke 4 gene was isolated and subcloned into pBluescript II. (^2)A restriction map of the 7.7-kb KpnI fragment was constructed, and small fragments from this region were subcloned into pBluescript II for sequencing. The details of the subcloning and sequencing strategy are depicted in Fig. 6. DNA sequencing was performed on double-stranded DNA by the dideoxy chain termination method, using the Sequenase Version II kit from U. S. Biochemical Corp.


Figure 6: Restriction map, subclones, and sequence strategy of genomic loci for Ke 6 and Ke 4 genes. a, horizontal line represents a 4.93-kb KpnI-BamHI genomic fragment containing both Ke 4 and Ke 6 genes. Vertical lines indicate restriction sites as noted above or below the lines. b, horizontal arrows indicate the direction and extent of sequencing. c, subclones generated from this region for sequencing purposes are denoted by horizontal lines. Restriction enzymes used to generate subclones are noted at the end of the lines, and size is indicated by the length of line. A 460-bp AccI-SmaI subclone used for the RNase protection experiment in Fig. 8is indicated by an asterisk and heavier line. d, direction of transcription of Ke 4 and Ke 6 genes is indicated by heavy horizontal arrows.




Figure 8: Ribonuclease protection anala, a 463-bp AccI-SmaI genomic subclone which extends 367 bp upstream of the first nucleotide in Ke 6a (clone 13.3.1) codon used for RNase protection experiment is depicted in the first line. The arrows indicate the direction and extent of transcripts generated from Ke 4 and Ke 6 genes corresponding to the sequence within this subclone. The subclone in pBluescript SK+ vector was linearized at BstEII or at StuI in order to synthesize antisense RNA probes directed by T3 polymerase. Lengths of RNA probes are indicated below. The shaded box in BstEII and StuI RNA probes indicates the region corresponding to the first exon of Ke 6 gene as determined from the cDNA clone of Ke 6a (13.3.1). The open box in the BstEII probe indicates the region corresponding to a 35-base region from the 3` end of the Ke 4 transcript. b, approximately 2.5 times 10^5 cpm of each probe was hybridized with 2 µg of poly(A) RNA isolated from mouse kidney or spleen. As controls, the same probes were hybridized with cRNA synthesized from Ke 6a cDNA (clone 13.3.1) or with tRNA ((-)RNA). After hybridization and RNase treatment the protected fragments were analyzed by electrophoresis on a 6% polyacrylamide gel containing 7 M urea and visualized by autoradiography. The arrowheads indicate the position of three protected bands of 57, 63, and 73 bp in kidney and spleen poly(A) RNA lanes corresponding to Ke 6 transcripts with both RNA probes. 35 bases of the protected RNA band corresponding to the 3` end Ke 4 mRNA is seen with the BstEII probe in kidney and spleen poly(A) RNA lanes.



Isolation and Sequencing of Ke 6b cDNA Clones

A cDNA 22A phage library was constructed with poly(A) RNA derived from mouse spleen using Life Technologies, Inc.'s superscript cDNA library synthesis kit. 10^6 recombinants were screened using the Ke 6a cDNA insert. Four positive clones were identified. The longest Ke 6b clone (0.9 kb) was subcloned into pBluescript SK+ plasmid for restriction mapping and complete sequence determination.

Ribonuclease Protection Analysis

A 460-bp genomic AccI-SmaI fragment, cloned into pBluescript SK+, was used for in vitro synthesis of two separate antisense RNA probes of 340 bases and 180 bases by truncating the insert used as a template. All probes were transcribed as directed using T3 RNA polymerase (Stratagene) in the presence of [-P]UTP. Approximately 2.5 times 10^5 cpm of each probe was hybridized with 2 µg of poly(A) RNA isolated from mouse kidney or mouse spleen. Hybridizations were carried out for 16 h at 50 °C in 40 mM PIPES, pH 6.4, 1 mM EDTA, pH 8.0, 0.4 M NaCl, and 80% formamide. RNA were digested with RNase A (40 µg/ml) and RNase T(1) (2 µg/ml) for 1 h at 30 °C, and the reactions were stopped by incubation with 300 µg/ml proteinase K in the presence of 0.5% SDS for 30 min at 37 °C(16) . Protected RNA fragments were analyzed by electrophoresis on a 6% polyacrylamide, 7 M urea gel and visualized by autoradiography. [-P]dATP-labeled HinfI-digested X174 DNA was used as a molecular weight marker.

Primer Extension Analysis

Primer extension analysis was performed using a 27-mer oligonucleotide complementary to position +27 to +1 of the Ke 6a cDNA. The primer was labeled at the 5` end with [-P]ATP and T4 polynucleotide kinase. The labeled oligonucleotide was cleaned on a superfine Sephadex G-50 spin column. 1.0 times 10^6 cpm of the primer oligonucleotide was hybridized to 4 µg of mouse kidney and spleen poly(A) RNA at 15 °C overnight in 10 µl of hybridization buffer (same buffer as that used for RNase protection assay). Extension was done using Superscript II reverse transcriptase at 42 °C in a 25-µl reaction. The extension products were analyzed using an 8% polyacrylamide-7 M urea sequencing gel along with a sequencing ladder using the same primer for the dideoxy sequencing.


RESULTS

Ke 6 Gene Expression Is Down-regulated in pcy Mice

The pcy mutation gives rise to a slowly progressing form of PKD (18) , similar to the jck model (19) and unlike the cpk model(20) , where the manifestation of the disease occurs soon after birth. Enlargement of kidneys is observed at about 8 weeks of age, although histological abnormalities are apparent even earlier, at day 1(21) . The pcy mutation is autosomal recessive and has been mapped to mouse chromosome 9(18) . Poly(A) RNA from kidney and liver of affected pcy, jck, cpk mice, and normal B6 mice was used for Northern blot analysis (Fig. 1). Ke 6a mRNA level is reduced to 54% in pcy/pcy kidneys compared with +/+ kidneys. Reduction in the level of Ke 6a mRNA in affected jck and cpk kidneys compared with +/+ kidney is 77 and 71%, respectively. This change in gene expression is specific for Ke 6, since there is no reduction in the level of actin, ferritin, and protein tyrosine phosphatase mRNAs(7, 8) . In the liver of the juvenile forms of PKD, the pcy and jck homozygotes, Ke 6 mRNA is lower by only 33 and 25%, respectively. However, in the congenital model of PKD, the cpk homozygote, there is a 68% reduction of Ke 6 mRNA compared with normal liver which we reported previously(7) . Since the liver of these murine PKD models is normal by histology and by morphology, it indicates that in kidney, the down-regulation of the Ke 6 gene must precede the onset of pathogenesis. In other words, Ke 6 must be causally rather than consequentially related to the molecular events leading to PKD(7) . By in situ hybridization and by immunofluorescent studies, Ke 6 mRNA has been localized to the proximal tubules and collecting ducts within the outer stripe of the kidney in normal animals(22, 23) . Earliest cystic changes in pcy mice, as well as in cpk and jck mice, are seen in proximal tubules and collecting ducts(18, 19, 20) . Occurrence of cysts in a region of kidney where the normal expression of the Ke 6 gene in these murine forms of PKD is repressed emphasizes a very important role of this enzyme in normal kidney structure and function.


Figure 1: Down-regulation of Ke 6 gene expression in murine models of PKD. 6 µg of poly(A) RNA from kidneys and liver of +/+, pcy/pcy,jck/jck, and cpk/cpk mice were used for Northern blot analysis. There was no gross morphologic difference in any organs besides kidney in homozygote pcy, jck or cpk mice. RNA was run on a 1% agarose, 2.2 M formaldehyde gel and blotted onto nylon membranes. [alpha-P]dCTP labeled Ke 6a cDNA was used as a probe for hybridization at 65 °C in Church's buffer for 16-20 h, and the membrane was washed in 0.1 times SSC, 0.1% SDS at 50 °C and exposed to x-ray film. Equal loading of RNA was confirmed by ethidium bromide staining intensity and by hybridizing the membrane with [alpha-P]dCTP-labeled 18 S cDNA probe.



Ke 6 Is a Single Copy Gene

Southern blot analysis indicates that the mouse genome contains a single copy of the Ke 6 gene. Fig. 2shows hybridization of P-labeled Ke 6a cDNA insert to mouse genomic DNA restricted with EcoRI, BamHI, HindIII, or PstI. Nucleotide sequence and restriction mapping of the Ke 6 genomic locus and Ke 6a and Ke 6b cDNAs reveal that, of these enzymes, only PstI cuts within the Ke 6 gene. The Ke 6a cDNA probe detects an 11-kb EcoRI fragment, a 4.9-kb BamHI fragment, and a 23-kb HindIII fragment as predicted by the restriction map of cosmid 32(24) . Three PstI fragments of sizes 1.5, 1, and 0.45 kb are detected in the genomic blot.


Figure 2: Ke 6 is a single copy gene. Genomic DNA isolated from C57Bl/J6 mouse was digested with BamHI, EcoRI, HindIII, and PstI and analyzed by Southern blotting. The membrane was hybridized with Ke 6a cDNA and washed in 0.2 times SSC, 0.1% SDS at 50 °C.



Cloning of the Ke 6b Transcript

The Ke 6 gene gives rise to two different mRNAs, Ke 6a and Ke 6b (Fig. 3). Ke 6a mRNA, which is about 1 kb, is abundant in liver and kidney and has been completely sequenced(7) . The Ke 6a mRNA is also found at very low levels in other tissues such as spleen, heart, and brain(7) . The Ke 6b transcript is estimated to be about 1.2 kb long from Northern gels and is present in spleen in moderate abundance (Fig. 3). In order to determine the nucleotide sequence of the Ke 6b transcript, a spleen cDNA library was constructed and screened using Ke 6a cDNA insert (clone 13.3.1). Four positive clones were isolated and subcloned into pBluescript vector. A 0.9-kb clone was restriction-mapped and determined to have an identical restriction pattern to the Ke 6a cDNA except for a 3` end BsmI fragment which was longer (Fig. 4). This clone was fully sequenced and revealed identical sequence homology to the Ke 6a transcript except for a 174-bp region in the latter third of the cDNA. The longer Ke 6b mRNA thus creates a Ke 6b protein whose carboxyl end differs from Ke 6a protein starting from residue 254 (Fig. 5).


Figure 3: Ke 6 gene gives rise to two transcripts. 4 µg of poly(A) RNA from a normal C57BL/6 mouse kidney and spleen was run on a formaldehyde-agarose gel and blotted for Northern analysis. The membrane was hybridized to Ke 6a cDNA and washed with 0.1 times SSC, 0.1% SDS at 50 °C.




Figure 4: Restriction map of Ke 6b cDNA reveals a longer BsmI fragment.




Figure 5: Comparison of the predicted amino acid sequence of Ke 6a and Ke 6b protein. The amino acid number of each protein is indicated in the beginning and at the end of each line.



Structure of Ke 6 Gene

The restriction map of a 4.93-kb D3 mouse genomic KpnI-BamHI fragment containing the entire Ke 6 gene and a part of the Ke 4 gene is shown in Fig. 6a. Smaller fragments from this piece of DNA were subcloned into pBluescript II vector for sequencing purposes using the strategy outlined in Fig. 6, b and c. A total of 4877 bp of sequence has been obtained which comprises the 3` end of the previously isolated Ke 4 gene and the entire Ke 6 gene (Fig. 7). The gene contains nine exons which range from 43 to 219 bp (Fig. 7, a and b). In Fig. 7b, a total of 1631 bp 5` to the ATG of Ke 6 gene and 194 bp 3` to the polyadenylation site is shown. Ke 6 gene is interrupted by 8 introns, all less than 200 bp in size. The exon-intron junctions are in accordance with consensus splice donor-acceptor patterns (GT-AG rule). In the open reading frames defined by Ke 6a and Ke 6b cDNAs, 5 codons are not split by intron-exon junctions (type 0), two of are type 1, where the intron-exon junction occurs after the first nucleotide of a codon, and one is type 2, where the intron-exon junction occurs after the second nucleotide of a codon. This trend is similar to that reported for vertebrate genes: 41% type 0, 36% type 1, and 23% type 2(25) . The poly(A) addition signal is at position 2015.


Figure 7: Intron-exon organization and complete nucleotide sequence of Ke 6 gene. a, open boxes represent Ke 6a exons, black box represents intron 8, and stippled boxes represent exons for the previously described Ke 4 gene. Numbers under the boxes indicate exon numbers for Ke 6a and Ke 6b transcripts. Heavy lines indicate relative lengths of Ke 6a and Ke 6b transcripts. b, exon sequences are represented in capital letters, introns and 3`- and 5`-flanking sequences are indicated in lowercase letters. Nucleotides are numbered in the right margin relative to +1 which marks the start of Ke 6a cDNA. Initiation of translation is proposed to occur at nucleotide +11. Amino acids and exon numbers are indicated in the right margin. The alcohol dehydrogenase superfamily signature motif is underlined. The Ke 6b mRNA-specific sequence is represented in bold letters. Codons which are split by exon-intron junctions are marked by a dot. The carboxyl-terminal Ke 6a amino acid number, which is different from Ke 6b due to alternative splicing, is presented within parenthesis. The poly(A) addition signal is italicized.



The alternative splicing pattern that gives rise to the spleen-specific Ke 6b transcript is due to failure of splicing of a 170-bp intron between exons 8 and 10 (Fig. 7). In contrast, the Ke 6a mRNA is created by excision of this intron which results in a smaller size message. This difference in splicing pattern is responsible for the deduced amino acid sequence difference between the two proteins at the carboxyl ends (Fig. 5). Within the Ke 6b-specific exon sequence (Fig. 7, boldface sequence) is a protein translational termination codon that gives rise to a longer 3`-untranslated region for the Ke 6b transcript (300 bases) compared with the 3`-untranslated region for the Ke 6a transcript (175 bases).

Putative Regulatory Elements in the Promoter Region

Analysis of the 5`-flanking nucleotide sequence of the Ke 6 gene shows the presence of several putative regulatory elements, which include NFkappab, Zeste, LBP-1, junB-US2, Pu-box, CAAT box, AP-2, and AP-3. At positions -361, -729, and -1142 there are GRE half-sites which may play a role in modulation of Ke 6 gene expression(8) . The consensus GRE motif is GGTACANNN-TGT(T/C)CT(26) . The second half of this palindrome is a hexameric sequence that is very well conserved and also an essential part of the GRE consensus. The first half of this palindrome is more variable(26) . The 11beta-hydroxysteroid dehydrogenase gene, which encodes an enzyme needed for the inactivation of corticosterone, also has GRE-like motifs in the promoter region(27) , similar to the Ke 6 gene. We have found that both of these genes are down-regulated in murine models of PKD(8) . It is likely this GRE-like hexamer is responsible for the coordinate regulation of steroid dehydrogenase genes in PKD. Interestingly, there are also three estrogen receptor half-sites (GGTCA) at positions +37, -134, and -247. The Ke 6 protein sequence also has high homology to pig 17beta-estradiol dehydrogenase (smallest Poisson probability = 4.9e and weaker homology to human 17beta-estradiol dehydrogenase (smallest Poisson probability = 0.35). The relevance of these putative steroid receptor binding sites remains to be determined.

Mapping the Transcription Initiation Site of the Ke 6 Gene

In order to map the transcription start site of the Ke 6 gene, RNase protection analysis was performed (Fig. 8). A 460-bp AccI-SmaI genomic fragment subcloned into pBluescript II SK+ containing the 3` end of the Ke 4 gene, the Ke 6 promoter region, and a portion of the first exon and first intron of the Ke 6 gene was used to synthesize two separate RNA probes, indicated in Fig. 8a. The BstII-digested template yields a 340-base probe containing only 35 bases of the Ke 4 exon sequence. The StuI-digested template yields a shorter probe of 180 bases, which contains no Ke 4 sequence. Both probes encode 57 bases of the Ke 6 exon as determined from the Ke 6a cDNA sequence (clone 13.3.1).

Approximately 2.5 times 10^5 cpm of probe was hybridized to in vitro transcribed Ke 6a cRNA (positive control), tRNA (negative control(-) RNA), kidney poly(A) RNA, and spleen poly(A) RNA for RNase protection assay. In all experiments, the positive control yields a major protected fragment of 57 bases, corresponding to the beginning of Ke 6a cDNA at position -10 (Fig. 8b, cRNA lane). The BstEII and StuI RNA probes yield common protected fragments of about 73, 63, and 57 bases, which represent Ke 6 transcripts having heterogeneous 5` ends. The previously cloned Ke 6a cDNA (13.3.1) represents a full-length clone whose 5` end corresponds to a start site at position -10 matching that represented by the 57-bp fragment. The 73- and 63-bp protected fragments represent mRNAs that are slightly longer than the 13.3.1 cDNA at position -16 and -26. The 35-bp protected fragment seen in kidney and spleen lanes with the BstEII-specific probe represents a portion protected by Ke 4 mRNA. Results of the RNase protection assay indicate that the Ke 6 gene has multiple start sites of transcription.

Primer extension analysis utilizing an antisense 27-mer complementary to bp +27 to +1 of the Ke 6a cDNA also revealed multiple transcription initiation sites, ranging from position -9 to -26 bp upstream of the predicted translational initiation codon (Fig. 9). The size range of the extension products are consistent with the result obtained with the RNase protection assay. However, with primer extension analysis the existence of more heterogeneity in the 5` end of Ke 6 mRNAs is apparent than that observed with RNase protection analysis. This could be because some of the mRNAs differ in size by 1 or 2 bp, which is resolved by sequencing gel analysis of the primer extension experiment and which may not be distinguishable with RNase protection assay. The three major bands seen in the primer extension analysis gel are at positions -9, -16, and -26. This phenomenon of multiple initiation sites of transcription is not uncommon in genes lacking a TATA sequence, since the TATA sequence plays a role in the accurate positioning of RNA polymerase II for the initiation of transcription(28, 29) .


Figure 9: Primer extension analysis. Poly(A) RNA (4 µg) from mouse kidney (K) and spleen (S) was hybridized with a 5` end-labeled oligonucleotide complementary to Ke 6a cDNA from +27 to +1. The primer was extended with reverse transcriptase and analyzed by electrophoresis on an 8% polyacrylamide, 7 M urea gel. Lanes A, C, G, and T, sequencing reaction was done using the same primer. The numbers on the right indicate the position of the extended products from the start of the initiator codon (+1) which is indicated in bold.



Similar results obtained by the above two method indicates that the Ke 6b mRNA is identical to the Ke 6a mRNA at the 5` end. This is further evidence that the two mRNAs differ only at the 3` end due to splicing differences within exon 8.


DISCUSSION

The Ke 6 gene is a single copy gene encoded within the mouse MHC on chromosome 17 identified by positional cloning techniques(7) . It is located approximately 25 kb telomeric to the class I H-2K gene. This region of the mouse chromosome 17, where the MHC and the t-complex region are located, make it an important region to identify new genes. The tcl-w5, an early acting embryonic lethal mutation within the t-complex region is recombinationally inseparable from the H-2K gene(30) . Therefore, the Ke 6 gene, in addition to other genes identified in this region, is a candidate for the tcl-w5 mutation. The search for new genes in the MHC has led to the discovery of several new genes which encode proteins of diverse functions. Human and mouse genes in the MHC were determined to be co-linear in their organization(31) . The Ke 6 gene, because of its positional similarity and 86% sequence homology to the Ring 2 gene, (^3)is most likely the mouse homolog of this human gene.

There is only a 234-bp gap between the 3` end of the previously identified Ke 4 gene (32) and the 5` end of the Ke 6 gene. Since both genes are transcribed in the same direction, it is possible that the span which encodes the 3`-untranslating region of the Ke 4 region encompasses the promoter region for Ke 6 gene. The close proximity of transcription units in this location of the MHC indicates that for some unapparent evolutionary benefit this region is highly enriched with genes.

The genomic organization of Ke 6 and other genes encoding steroid metabolic enzymes are not similar to each other. Human genes for the 11beta-hydroxysteroid dehydrogenase(27) , 3beta-hydroxysteroid dehydrogenase/Delta isomerase(33) , and 17beta-hydroxysteroid dehydrogenase (34) which have been cloned all have unrelated exon/intron organization and also all vary in length.

We report that the Ke 6 gene is down-regulated in a third murine model of PKD: the pcy mouse (Fig. 1). Thus far, Ke 6 is repressed in all murine models of PKD that we have investigated: cpk, jck(7) , and pcy mice (this report). This suggests that Ke 6 has a critical role in the normal functioning of the kidney and that molecular events leading to its repression in kidney may be responsible for the development of renal cysts. In the cpk homozygote, the Ke 6 gene is down-regulated to the same extent in the liver as in the kidney, even though the liver of affected animals appears perfectly normal by morphology and by histological examination(7) . The repression of Ke 6 in liver suggest that an aberration in the regulation of expression of Ke 6 gene in the kidney does not occur as a consequence of a diseased organ, but rather it occurs before the onset of pathogenesis(7) . The Ke 6a protein sequence is homologous to bacterial and mammalian steroid dehydrogenases and also to human prostaglandin dehydrogenase (8) . Similarity of Ke 6a protein to the dehydrogenases is high within specific functional domains of these enzymes: the NAD binding domain and the dehydrogenase active site domain. Furthermore, we find that the rat 11beta-hydroxysteroid dehydrogenase has a similar pattern of aberrant regulation as the Ke 6 gene in older cpk heterozygote liver and in young cpk homozygote liver(8) . Therefore the structural homology and regulatory expression pattern similarity to a known steroid dehydrogenase strongly suggest that Ke 6 itself is a steroid dehydrogenase(8) . Incidentally, the gene for a steroidogenic enzyme, the 21-steroid hydroxylase gene, is also encoded within the MHC.


FOOTNOTES

*
This work was supported by NIDDK Grant DK 48098 and March of Dimes Birth Defects Foundation grant (to N. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34072[GenBank].

§
Current address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

To whom correspondence should be addressed: Children's Hospital, Nephrology Division, Hunnewell-3, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7756; Fax: 617-730-0435.

(^1)
The abbreviations used are: PKD, polycystic kidney disease; kb, kilobase pair(s); bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; MHC, major histocompatibility complex.

(^2)
B. St.-Jacques, unpublished data.

(^3)
J. Trowsdale, personal communication.


ACKNOWLEDGEMENTS

We thank David Woo (UCLA) for providing pcy mouse tissue and David Beier (Brigham and Women's Hospital) for providing jck mouse tissues.


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