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The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.

Hedgehog (HH) signaling is one of the key pathways with major significance for embryogenesis, tumorigenesis, and stem cell maintenance. Glioma-associated oncogene 1 (GLI1) is a transcription factor that acts as the terminal signaling effector but also represents a pathway target gene. Here we report the identification and functional properties of novel GLI1 splice variants generated by skipping exons 2 and 3 and encoding an N-terminal truncated GLI1 protein (GLI1DeltaN). Analysis of the GLI1DeltaN mRNAs in adult human tissues revealed comparable expression levels to the full-length GLI1 (GLI1FL), whereas in tumor cell lines a generally lower and more variable expression pattern was observed. Furthermore, GLI1DeltaN is up-regulated by HH signaling to the same extent as GLI1FL but has a weaker capacity to activate transcription. However, in specific cellular contexts GLI1DeltaN may be more potent than GLI1FL in activating endogenous gene expression. Moreover, the dual-specificity tyrosine phosphorylation-regulated kinase 1 (Dyrk1) potentiates the transcriptional activity of GLI1FL but not GLI1DeltaN. Interestingly, GLI1FL, in contrast to GLI1DeltaN, is localized solely at the nucleus, in line with its increased transcriptional capacity. The negative regulator of the pathway, Suppressor of Fused (SUFU), elicits a cytoplasmic retention of the GLI1 isoforms, which is more pronounced for GLI1FL, as this contains an N-terminal SUFU binding domain. Collectively, our findings reveal that the activation mechanism of the terminal transducer of the pathway, GLI1, is mediated not only by GLI1FL but also by the GLI1DeltaN variant.

Patched1 (PTCH1) is one of the key molecules involved in the Hedgehog (HH) signaling pathway and acts as the receptor of HH ligands. Additionally, PTCH1 inhibits the positive signal transductor Smoothened (SMO). Several PTCH1 splice variants are known but the functional differences among them are not clear. Here, we demonstrate the unique biological properties of the PTCH1 isoforms generated by alternative first exon usage. All isoforms examined worked as functional receptors of both Sonic HH and Desert HH. However, the signaling upregulated isoforms PTCH1-1B and -1C inhibited SMO and the pathway transcription factors glioma 1 (GLI1) and GLI2 to a higher extent than PTCH1-1 and -1Ckid. Moreover, in situ hybridizations allowed the detection of the Ptch1 isoforms in specific structures of the developing mouse embryo. Additionally, the differences in the N-terminal tail had a dramatic influence on the steady states of the proteins, with PTCH1-1B and -1C levels being significantly higher than PTCH1-1 and -1Ckid. This implies that the pronounced signaling inhibitory properties of PTCH1-1B and -1C may be mostly due to this high-protein expression rather than to intrinsic functional differences. Thus, our study supports a role of splicing variation and promoter choice for HH signaling regulation.

The mouse Cyp3a locus on chromosome 5 was analyzed by the use of bacterial artificial chromosomes. Five out of the six known Cyp3a genes, Cyp3a11, Cyp3a13, Cyp3a16, Cyp3a25, Cyp3a41 and Cyp3a44 were found to be linked to each other, however, Cyp3a13, possibly because of a distant position from the main body of the locus was not. In the intergenic regions additional Cyp3a genomic sequences were identified providing evidence for duplication events within the locus. Moreover, a Cyp3a41 duplicated gene, Cyp3a41A, is expressed at significant levels as the corresponding mRNA can be detected in the EST database. In line, therefore, with the higher number of mouse versus human CYP3As, the mouse locus covers a larger chromosomal region and appears to be considerably more complex.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

Microsatellite markers assigned to chromosome 9q22.3-q31 were used in order to analyze squamous cell carcinomas from human skin. From the informative samples examined, one showed loss of heterozygosity with both microsatellite markers D9S109 and D9S180. Furthermore, in four cases with D9S109 and in one case with both microsatellite markers, genetic instability was observed in the tumors. These results are interpreted as suggestive of two distinct genetic changes associated with skin squamous cell carcinomas: Loss of a tumor suppressor gene that might relate to the observed frequent loss of a co-localized gene in basal cell carcinomas and alteration of replication/mismatch-repair factors, in similarity to the ones thought to be involved in the genetic instability detected in hereditary nonpolyposis colorectal cancer.

Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2.To search for mutations and genomic instability in sporadic and inherited BCCs.DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database.Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g.2885GC in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g.2839TG in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1.Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

Background Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. Results We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. Conclusions Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.

The male-specific CYP2C13 gene has been isolated from two independent rat genomic libraries. This gene spans more than 50 kb and contains eight introns which are subject to the GT-AG rule. Two allelic forms of the CYP2C13 gene were identified. Determination of the exonic sequences revealed that one of them encodes cytochrome P450(+g) and the other encodes cytochrome P450(-g). Using allele-specific restriction enzyme sites, a good correlation between the genotype and the phenotype of CYP2C13 was shown. Nucleotide substitutions between the (+g) and the (-g) genes exist not only in the exons but also in the introns and the 5'-flanking region. Although five nucleotide differences were identified within 287 base pairs of the (+g) and (-g) 5'-flanking regions, the transcription initiation sites were identical. In addition to a canonical TATA box located 31 base pairs upstream of the start site of transcription, putative binding sites for the liver-enriched and liver-specific transcription factors HNF1/LF-B1/APF, HNF3, HNF4/AF-1, C/EBP, LAP, and eH-TF/TGT3 and the ubiquitous factors NF-1 and OTF-1 were identified.

A novel member of the cytochrome P450 2C gene subfamily was identified by screening rat prostate cDNA libraries. Two independent clones were isolated. Clone pros1 was 1031 bp long and contained a bizarre replacement in place of putative exon 1. Clone pros2 was 1755 bp long, contained a complete 3' end, and also had bizarre sequences in place of exon 1, which in this case were compatible with an unspliced intron. Northern analysis revealed mRNA expression in the liver and the kidney. Interestingly, although livers of mature rats of both sexes have comparable amounts of P4502C24 mRNA, a dramatic sex difference is seen in the kidney where only males express detectable levels of this mRNA.

To isolate cDNAs for forms of cytochrome P450 from rat prostate, a lambda gt11 cDNA library from this tissue was screened with a mixture of oligonucleotide probes directed against the conserved heme binding region of different P450 isozymes. A cDNA clone (PP1) encoding a part of a novel form of cytochrome P450 was isolated and the deduced amino acid sequence showed 76% identity with cytochrome P450 IVA1, indicating that PP1 is a member of the same subfamily. Northern blot analysis of total RNA from prostates of untreated rats revealed that two mRNAs of approximately 2.8 and 2.2 kb hybridize to PP1. The level of mRNA was induced fivefold above the level in intact animals by androgen treatment of castrated rats. Analysis of poly(A)+RNA levels in different tissues on Northern blots showed high constitutive expression of PP1 in the kidney, but no signal was detectable with RNA from liver; a weak signal was detected in the retina. Subsequent screening of a rat kidney cDNA library led to the isolation of the full-length clone KP1, which differs from Pp1 only in three nucleotide positions. KP1 is 1,957 bp long and contains a 1,527-bp-long open reading frame encoding a protein of 508 amino acids. In situ hybridization of rat kidney sections with PP1 showed that this P450 form is expressed in the outer stripe of the outer medulla, indicating its localization in the proximal tubules.

Cytochrome P450 15 beta is a female-specific gene product that catalyzes the hydroxylation of steroid sulfates at the 15 beta position. Isolation and analysis of the gene for rat P450 15 beta reveals nine coding exons and encompasses more than 35 kb of chromosomal DNA. The intron-exon junctions are at similar positions with the P450 genes of the II family, whose gene structure has been determined. Sequencing of about 2 kb of the 5'-flanking region indicates the presence of the Alu-like R.dre.1 repetitive sequence, a GA-rich stretch that is also found in the 5'-flanking DNA of the male-specific cytochrome P450 16 alpha gene, the enhancer octamer sequence ATGCAAAT, and three CAAAGTT repeats just upstream from the TATA box. Primer extension reveals a major and a minor transcription start site located 22 and 26 bases 5' to the translation initiation codon, respectively. This gene is developmentally regulated and transcriptional activation accounts, at least partly, for the sexually differentiated expression of cytochrome P450 15 beta.

Patched homolog 1 (PTCH1) is a key component of the Hedgehog (HH) signaling pathway with three alternative first exons, but only exon 1B transcription depending on HH activation. Here, we show that in both human and mouse a novel PTCH1 first exon (1C) is expressed. Exon 1C transcription is upregulated by HH signaling, but the resulting PTCH1-1C protein has a lower capacity for pathway inhibition than PTCH1-1B.

The PTCH1 gene is a human tumour suppressor gene frequently mutated in basal cell carcinoma (BCC) and several other tumour types. It encodes a receptor for soluble factors of the hedgehog family. Binding of hedgehog to the receptor relieves its inhibitory action on the transmembrane co-receptor Smoh. In this study we describe alternative first exons of the PTCH1 tumour suppressor gene and show that they are differentially regulated in normal tissues, exon 1B being expressed at very low levels and the major mRNA species containing exon 1 or 1A. Exon 1B transcripts were found to be specifically upregulated in nodular BCCs. The different PTCH1 transcripts all encode proteins that interact with Smoh in doubly transfected cells. Furthermore, functional assays demonstrated that whereas all PTCH1 isoforms can inhibit the activity of SHH, only the PTCH1B isoform is capable of fully inhibiting Smoh activity. The results indicate that in tumour cells the PTCH1B promoter is specifically activated and importantly, that the N-terminal part of PTCH1 including exon 1B is required for full inhibition of Smoh signaling but not for physical interaction with Smoh.

The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43 . Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli , and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4–3A7 and 3A7–3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3′ end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.

The cytochrome P-450 2C18 gene was found by reverse transcription-PCR to represent the most abundantly expressed gene of the P-450 2C subfamily in human epidermis. However, in addition to the canonical mRNA of nine exons, transcripts that have skipped exon 4 or 5, exons 4, 5, and 6, or exons 4, 5, 6, and 7 were also identified in this tissue. Remarkably, circular RNA transcripts synthesized by the joining of the donor and acceptor splice sites of the same exon were detected in human epidermis for exons 4 and 5. Moreover, molecules composed of exons 4, 5, and 6 with the donor splice site of exon 6 joined to the acceptor splice site of exon 4 or composed of exons 4, 5, 6, and 7 with the donor splice site of exon 7 joined to the acceptor splice site of exon 4 were also found to be present in this tissue. In rat testis, a similar analysis allowed the detection of a circular RNA molecule composed of exons 6 and 7 of the androgen binding protein (ABP) gene, with the donor splice site of exon 7 joined to the acceptor splice site of exon 6, and of an ABP mRNA which had skipped exons 6 and 7. These results apparently substantiate the hypothesis that alternative pre-mRNA splicing has the potential to generate not only mRNAs that lack one or more exons but also circular RNA molecules that are composed of the exons that are skipped. However, additional 2C18 circular species containing various combinations of exons were also detected in human epidermis, and an exon 6-skipped ABP mRNA molecule was identified in rat testis. This observation is interpreted as indicative that at low frequency, numerous circular RNA formation and exon skipping events may occur, allowing the joining of a variety of different combinations of exons. Moreover, the relative stability of these molecules is apparently the key factor that determines the relative ease of their detection.

To identify expressed sequences from the candidate genomic region of the nevoid basal cell carcinoma syndrome (9q22.3), the CpG island rescue PCR methodology was employed on the yeast artificial chromosome (YAC) ICI-8AD8 that contains microsatellite marker D9S180. A positive clone (IR10, size 350 bp) was isolated by screening a human epidermal cDNA library with the island rescue PCR products and mapped back to the 9q22.3 region. To obtain additional sequence information from IR10, the rapid amplification of cDNA ends (RACE) methodology was employed. Within the longest IR10 RACE product, two segments having similarities with coronin, a G-like and actin-binding protein of Dictyostelium discoideum, as well as with p57, a recently cloned human actin-binding protein, were revealed. In fact, these two segments have properties of typical exons, suggesting that the RACE products and clone IR10 represent unspliced pre-mRNAs. Using the RACE methodology with primers originating from these two exons, it was shown that they splice together and the sequences corresponding to the processed IR10 mRNA were revealed. This mRNA maps back to 9q22.3 and furthermore was found to code for a novel protein composed of 525 amino acids and containing five WD repeats. The presence of the WD repeat motif, considered to function in protein-protein interactions, implies that the new protein might have a role in intracellular signaling. Moreover, Northern analysis indicated that, in addition to epidermis, the brain is a tissue with relatively high levels of expression of this gene. Finally, Southern analysis showed a functional conservation of the WD repeat protein in various species.

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

The reverse transcriptase (RT) / polymerase chain reaction (PCR) methodology was used with a set of primers that corresponds to two conserved regions of the cytochrome P450s of subfamily 2C, in order to identify the members of this group of enzymes that are expressed at the RNA level in rat brain. Seven RT/PCR clones were sequenced from female brain, and four were found to code for P450 2C7 while three for P450 2C12. Using the same methodology nine RT/PCR clones were sequenced from olfactory lobes of ethanol treated male rats and three were found to code for P450 2C6, one for P450 2C11, three for P450 2C12 and two for P450 2C23. Neither ethanol administration nor hypophysectomy or growth hormone treatment had significant effects on the expression levels of these cytochromes in the brain.

The Rapid Amplification of cDNA Ends (RACE) procedure was used in order to investigate the heterogeneity of the 5′ region of the cytochrome P450 2C24 transcripts. This methodology revealed that in male kidney sequences typical of an exon 1 predominate. In the prostate though, instead of exon 1, several sequences indicative of alternative splicing events and possibly differential promoter usage were identified. Furthermore, both in prostate and kidney, transcripts having upstream of exon 2, exon 4 sequences were also observed. This implies that either the 2C24 gene is a nontypical P450 gene having exon 4 sequences both upstream and downstream of exon 2 or that trans splicing of canonical P450 2C24 primary transcripts occurs in these tissues.

Complementary DNA clones encoding the male-specific rat liver cytochrome P-450 g have been isolated by cross-hybridization with sequences from the female-specific rat liver cytochrome P-450 15 beta. Tissue distribution analysis indicates the liver as the organ with major expression of this cytochrome P-450 gene. Minimal P-450 g expression was also detected in prostate, kidney, heart, and brain. A developmental analysis reveals liver expression in the 8-week-old male and to a lesser extent in the 4-week-old male, but no detectable expression is seen in females of these ages or in 1- and 2-week-old rats from both sexes. Hypophysectomy of female rats dramatically increases hepatic expression of P-450 g, whereas continuous GH administration represses hepatic expression in male or female hypophysectomized rats. In similarity to P-450 15 beta and P-450 16 alpha, therefore, the cytochrome P-450 g gene in liver is GH regulated.

cDNA clones for P-45016α were isolated from a male liver λgt 11 expression library using antibodies against P-45016α. The clones encompassed 1633 and 1791 bp, respectively. The latter clone contained the whole coding sequence. The 20 deduced NH2-terminal amino acids were identical to those of P-450h and the cDNA sequence was in complete agreement with that of P-450 (M-1). Northern blots showed that P-45016α in the rat liver is pretranslationally regulated by the growth hormone secretory pattern. Southern blots indicated that few genes belong to the same P-450 gene family as P-45016α.

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

The cytochrome P450 2C (CYP2C) gene locus was found to include a novel exon 1 sequence with high similarity to the canonical exon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) and PCR amplifications of human liver cDNA revealed the presence of several intergenic species containing the CYP2C18 exon 1-like sequence spliced to different combinations of exonic and intronic sequences from the CYP2C9 gene. One splice variant was found to have an open reading frame starting at the canonical translation initiation codon of the CYP2C18 exon 1-like sequence. Another variant consisted of the nine typical CYP2C9 exons spliced after the CYP2C18 exon 1-like sequence through a segment of CYP2C9 5' flanking sequences. Moreover, analysis of bacterial artificial chromosome (BAC) clones revealed that the CYP2C18 exon 1-like sequence was located in the intergenic region between the CYP2C19 and CYP2C9 genes. The finding that a solitary exon is spliced with sequences from a neighboring gene may be interpreted as representing a general evolutionary mechanism aimed at using the full expression potential of a cell's genomic informational content.

In order to identify cytochrome P450s that are expressed in porcine ovaries, the RT/PCR methodology was used with a see of primers that corresponds to two conserved regions of the 2C gene subfamily of these enzymes. Five independent cDNA clones were isolated from preovulatory follicles (PF1, PF11, PF13, PF14, PF15) and six from the corpus luteum (CL1, CL6, CL7, CL8, CL12, CL13). The structural identities categorize these 11 P450s into two groups, group A consisting of PF1, CL7 and CL8 and group B consisting of the remaining clones. In addition, segments that are apparently interchanged between the clones were also revealed, implicating gene conversion events that have homogenized the sequence of these P450s. Furthermore, although the larger group of these porcine enzymes (group B forms) are structurally very similar to the known human P450s of the 2C gene subfamily, the group A forms are much more distantly related, implying that the 2C P450s might have evolved differently in the two species.