Unique Gene Structure Research Articles

Differential Regulation of MicroRNA Expression In Polycythemia Vera CD34+ Cells

AbstractAbstract 4785 Introduction:Polycythemia vera (PV) is a clonal stem cell disorder characterized by unregulated red cell, white cell and platelet production, a phenotype attributable to an activating point mutation (V617F) in JAK2, a tyrosine kinase utilized for signaling by Type 1 hematopoietic growth factor receptors. However, it is generally conceded that JAK2 V617F is not the initiating mutation in PV, nor are any of the other gene mutations, deletions and amplifications discovered to date. Deregulated gene transcription is also a feature of PV but a unified mechanism for this also cannot be ascribed to the currently known genetic defects. MicroRNAs (miRs) are non-coding 18–22nt RNAs that function as posttranscriptional gene repressors by binding to complementary target sequences in the mRNA 3′UTR to inhibit protein translation. There is growing evidence that miRs regulate hematopoiesis not only at the committed hematopoietic progenitor cell level but also at the level of the hematopoietic stem cell (HSC). Studies of miR behavior in PV have documented differential expression during erythroid differentiation as well as differential expression in granulocytes and platelets. However, PV is a stem cell disorder and to date there has been no analysis of miR behavior in PV HSC. We report here differential miR expression in PV peripheral blood (pb) CD34+ cells. Methods:We studied pb CD34+ cells from eight PV patients (four females and four males, all JAK2 V617F-positive) and six normal donors (three females and three males) in conjunction with a study of mRNA expression in PV pb CD34+ cells by oligonucleotide microarray. Using total cell RNA, microRNA expression was analyzed with the Agilent Human microRNA Microarray Kit Version 3, which contains probes for 866 human miRs. The TaqMan MicroRNA Assay (Applied Biosystems) was used for quantitative reverse-transcription PCR to verify differential miR expression. The Student unpaired t test was used for statistical analysis and P values £ 0.05 were considered significant. Results:Overall, in the 8 PV patients as a group, there were 130 positive calls with differential regulation of 81 miRs (p <0.05) (23 up regulated and 58 down regulated) and there was no correlation between miR expression and the JAK2 V617F allele burden. Given the phenotypic and genotypic differences between men and women PV patients, we analyzed differential miR expression separately by gender to identify gender-specific effects. 75 miRs (26 up and 49 down regulated) were differentially expressed in the male PV patients and 32 (7 up and 25 down regulated) in the women, confirming similar observations of gender-related differences in global mRNA expression in PV pb CD34+ cells. Amongst the differentially regulated miRs in the group as a whole were the miR-H1 group, miR-125a-3p and 196b. The miR-H1 group, also known as miR-17-92, is an oncomir with a unique gene structure from which one primary transcript yields six distinct miRs. This cluster was only up regulated in male PV patients. Likewise, miR-125a-3p was also up regulated only in males. Its expression is dependent on DICER, a gene also only up regulated in male patients. MiR-125a-3p has been identified as being preferentially expressed in HSC, acts to expand HSCs by preventing apoptosis and favors a myeloid fate. MiR-196b, which was concordantly down regulated in both men and women, is normally up regulated in HSC. Altogether, consistent with their different phenotypes, 31 miRs were differently regulated solely in the male patients (16 up regulated and 15 down), while only 12 miRs were differently regulated solely in the women (2 up and 10 down regulated) Seven miRs were concordantly up (miR-575) or down regulated (miR-150; miR-181c; miR-196b; miR-551b; miR-769-5p and miR-874) by both the men and women patients. Notably, down regulation of miR-150 at the HSC level is consistent with its role in lineage-specific progenitor cell commitment. Since there appears to be little overlap with respect to target genes for this core group of miRs, multiple venues for translational control are likely to be involved in the pathogenesis of PV. Conclusion:These data establish the presence of differential miR expression in PV HSC and provide insight into potential mechanisms for PV HSC expansion, confirm a genetic basis for the gender-specific differences that characterize PV and identify novel therapeutic targets for drug discovery. Disclosures:No relevant conflicts of interest to declare.

Combining Comparative Sequence and Genomic Data to Ascertain Phylogenetic Relationships and Explore the Evolution of the Large GDSL-Lipase Family in Land Plants

The GDSL-lipase gene family is a very large subfamily within the supergene family of SGNH esterases, defined by the distinct GDSL amino acid motif and several highly conserved domains. Plants retain a large number of GDSL-lipases indicating that they have acquired important functions. Yet, in planta functions have been demonstrated for only a few GDSL-lipases from diverse species. Considering that orthologs often retain equivalent functions, we determined the phylogenetic relationships between GDSL-lipases from genome-sequenced species representing bryophytes, gymnosperms, monocots, and eudicots. An unrooted phylogenetic tree was constructed from the amino acid sequences of 604 GDSL-lipases from seven species. The topology of the tree depicts two major and one minor subfamily. This division is also supported by the unique gene structure of each subfamily. Because GDSL-lipase genes of all species are present in each of the three subfamilies, we conclude that the last common ancestor of the land plants already possessed at least one ancestral GDSL-lipase gene of each subfamily. Combined gene structure and synteny analyses revealed events of segmental duplications, gene transposition, and gene degeneration in the evolution of the GDSL-lipase gene family. Furthermore, these analyses showed that independent events of intron gain and loss also contributed to the extant repertoire of the GDSL-lipase gene family. Our findings suggest that underlying many of the intron losses was a spliceosomal-mediated mechanism followed by gene conversion. Sorting the phylogenetic relationships among the members of the GDSL-lipase gene family, as depicted by the tree and supported by synteny analyses, provides a framework for extrapolation of demonstrated functional data to GDSL-lipases, whose function is yet unknown. Furthermore, function(s) associated with specific lineage(s)-enriched branches may reveal correlations between acquired and/or lost functions and speciation.

Mir-17-92, a cluster of miRNAs in the midst of the cancer network

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs (ncRNAs) that function to regulate gene expression at the post-transcriptional level. Although their functions were originally described during normal development, miRNAs have emerged as integral components of the oncogenic and tumor suppressor network, regulating nearly all cellular processes altered during tumor formation. In particular, mir-17-92, a miRNA polycistron also known as oncomir-1, is among the most potent oncogenic miRNAs. Genomic amplification and elevated expression of mir-17-92 were both found in several human B-cell lymphomas, and its enforced expression exhibits strong tumorigenic activity in multiple mouse tumor models. mir-17-92 carries out pleiotropic functions during both normal development and malignant transformation, as it acts to promote proliferation, inhibit differentiation, increase angiogenesis, and sustain cell survival. Unlike most protein coding genes, mir-17-92 is a polycistronic miRNA cluster that contains multiple miRNA components, each of which has a potential to regulate hundreds of target mRNAs. This unique gene structure of mir-17-92 may underlie the molecular basis for its pleiotropic functions in a cell type- and context-dependent manner. Here we review the recent literature on the functional studies of mir-17-92 and highlight its potential impacts on the oncogene network. These findings on mir-17-92 indicate that miRNAs are integrated components of the molecular pathways that regulate tumor development and tumor maintenance.

Gene Structure Induced Epigenetic Modifications of pericarp color1 Alleles of Maize Result in Tissue-Specific Mosaicism

BackgroundThe pericarp color1 (p1) gene encodes for a myb-homologous protein that regulates the biosynthesis of brick-red flavonoid pigments called phlobahpenes. The pattern of pigmentation on the pericarp and cob glumes depends upon the allelic constitution at the p1 locus. p1 alleles have unique gene structure and copy number which have been proposed to influence the epigenetic regulation of tissue-specific gene expression. For example, the presence of tandem-repeats has been correlated with the suppression of pericarp pigmentation though a mechanism associated with increased DNA methylation.Methodology/Principal FindingsHerein, we extensively characterize a p1 allele called P1-mosaic (P1-mm) that has mosaic pericarp and light pink or colorless cob glumes pigmentation. Relative to the P1-wr (white pericarp and red cob glumes), we show that the tandem repeats of P1-mm have a modified gene structure containing a reduced number of repeats. The P1-mm has reduced DNA methylation at a distal enhancer and elevated DNA methylation downstream of the transcription start site.Conclusions/SignificanceMosaic gene expression occurs in many eukaryotes. Herein we use maize p1 gene as model system to provide further insight about the mechanisms that govern expression mosaicism. We suggest that the gene structure of P1-mm is modified in some of its tandem gene repeats. It is known that repeated genes are susceptible to chromatin-mediated regulation of gene expression. We discuss how the modification to the tandem repeats of P1-mm may have disrupted the epigenetic mechanisms that stably confer tissue-specific expression.

Hypoxia‐inducedBNIP3expression and mitophagy:in vivocomparison of the rat and the hypoxia‐tolerant mole rat,Spalax ehrenbergi

The blind subterranean mole rat of the Spalax ehrenbergi superspecies is an excellent animal model for hypoxic tolerance. Unique physiological, functional, and gene structure changes allow Spalax species to survive lower oxygen levels than most terrestrial animals. BNIP3, an HIF-1 dependent hypoxia-response gene, has a proapoptotic function; however, expression is suppressed in many types of cancers. Under hypoxic conditions, BNIP3 also functions as a mediator of mitochondrial autophagy, a survival adaptation to control ROS production and DNA damage. Using real-time PCR and Western blotting, we investigated the impact of hypoxia on BNIP3 expression and mitophagy, in the skeletal muscle and heart, of the Rattus and two Spalax species. BNIP3 transcript, as well as protein levels, increased to significantly higher levels under hypoxia in Rattus tissues, with smaller changes in Spalax. Mitophagy was correlated with BNIP3 expression in the heart with an inverse correlation to hypoxia tolerance. A dense network of vessels in Spalax muscle may offer protection from physiological hypoxia, while the response in Rattus reflects the increase of hypoxic stress. In Spalax tissues, as in many cancers, BNIP3 expression and mitophagy are significantly less affected by hypoxia. Similar mechanisms, beneficial to organisms adapted to stressful environments, may also confer malignant cells with survival features. Understanding the molecular basis of such adaptations may enhance development of new therapeutic modalities.

Phylogenomic Relationships of Rice Oxalate Oxidases to the Cupin Superfamily and Their Association with Disease Resistance QTL

Abstract Rice oxalate oxidase genes (OXO) may play a role in resistance to Magnaporthe oryzae. Genome analyses showed four tandemly duplicated OXO genes, OsOXO1–OsOXO4, which mapped to a blast resistance QTL in chromosome 3. These genes have &gt;90% nucleotide and amino acid identity, but they have unique gene structures, conserved motifs, and phylogeny compared to the 70 other members of the cupin superfamily in the Nipponbare genome, which were divided into several classes. In resistant and susceptible Vandana/Moroberekan advanced backcross lines, only OsOXO4 was expressed during rice–M. oryzae interactions, and its expression increased earlier in resistant than susceptible lines. The earlier expression of OsOXO4 in resistant lines correlated with a 26-bp promoter insertion containing an additional copy of the bacterial responsive nodulation cis-element. Our results showed that OsOXO1–4 are in a separate class of rice cupin genes and supports a role for the promoter variant of OsOXO4 in resistance to M. oryzae.

Production of serotype C specific and serotype C/D generic monoclonal antibodies using recombinant H C and H N fragments from Clostridium botulinum neurotoxin types C 1 and D

Sequence variability of Clostridium botulinum serotypes C and D is particularly complex. Some serotype C and D strains have unique gene structures that encode mosaic isoforms of botulinum neurotoxin (BoNT) containing components of both BoNT type C 1 (BoNT/C 1) and BoNT type D (BoNT/D). Such sequence variability and the potential for cross neutralisation must be taken into consideration when developing serotype C and D detection and identification assays. Three fusion proteins containing either a fragment from the carboxyl-terminal domain of the heavy chain (H C) of BoNT/C 1 (strain 573), a fragment from the H C of BoNT/D (strain BVD/-3) or a fragment from the amino-terminal domain of the heavy chain (H N) of BoNT/C 1 (strain 573) were expressed in Escherichia coli, and administered as immunogens to mice. Monoclonal antibodies (mAbs) against the recombinant BoNT fragments were prepared by three fusions. MAbs recognising native BoNT/C 1 and BoNT/D were detected by enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies (mAbs) were produced, six of which recognised a BoNT fragment that is highly conserved across all serotype C and D producing strains. We conclude that these mAbs and this approach to mAb production may facilitate the development of immunological diagnostic techniques that are not constrained by the existence of mosaic isoforms for the detection and identification of serotypes C and D.

Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis

Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination. We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients. We examined 24 Japanese AD patients with "FLG-shotgun" method. Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients. We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.

The PIP and TIP aquaporins in wheat form a large and diverse family with unique gene structures and functionally important features

Aquaporins, members of major intrinsic proteins (MIPs), transport water across cellular membranes and play vital roles in all organisms. Adversities such as drought, salinity, or chilling affect water uptake and transport, and numerous plant MIPs are reported to be differentially regulated under such stresses. However, MIP genes have been not yet been characterized in wheat, the largest cereal crop. We have identified 24 PIP and 11 TIP aquaporin genes from wheat by gene isolation and database searches. They vary extensively in lengths, numbers, and sequences of exons and introns, and sequences and cellular locations of predicted proteins, but the intron positions (if present) are characteristic. The putative PIP proteins show a high degree of conservation of signature sequences or residues for membrane integration, water transport, and regulation. The TIPs are more diverse, some with potential for water transport and others with various selectivity filters including a new combination. Most genes appear to be expressed as expressed sequence tags, while two are likely pseudogenes. Many of the genes are highly identical to rice but some are unique, and many correspond to genes that show differential expression under salinity and/or drought. The results provide extensive information for functional studies and developing markers for stress tolerance.

Evolution of the Cytoplasmic and Mitochondrial Phosphagen Kinases Unique to Annelid Groups

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known-cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.

Increased Leptin Expression in Common Carp (Cyprinus carpio) after Food Intake But Not after Fasting or Feeding to Satiation

Leptin is a key factor in the regulation of food intake and is an important factor in the pathophysiology of obesity. However, more than a decade after the discovery of leptin in mouse, information regarding leptin in any nonmammalian species is still scant. We report the identification of duplicate leptin genes in common carp (Cyprinus carpio). The unique gene structure, the conservation of both cysteines that form leptin's single disulfide bridge, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and carp leptins, despite low amino acid identity. The liver is a major yet not the only site of leptin expression. However, neither 6 d nor 6 wk of fasting nor subsequent refeeding affected hepatic leptin expression, although the carp predictably shifted from carbohydrate to lipid metabolism. Animals that were fed to satiation grew twice as fast as controls; however, they did not show increased leptin expression at the termination of the study. Hepatic leptin expression did, however, display an acute and transient postprandial increase that follows the postprandial plasma glucose peak. In summary, leptin mRNA expression in carp changes acutely after food intake, but involvement of leptin in the long-term regulation of food intake and energy metabolism was not evident from fasting for days or weeks or long-term feeding to satiation. These are the first data on the regulation of leptin expression in any nonmammalian species.

Unique gene structure and paralogy define the 7D-cadherin family.

Cadherins are Ca2+-dependent transmembrane glycoproteins crucial for cell-cell adhesion in vertebrates and invertebrates. Classification of this superfamily due to their phylogenetic relationship is currently restricted to three major subfamilies: classical, desmosomal and protocadherins. Here we report evidence for a common phylogenetic origin of the kidney-specific Ksp- (Cdh16) and the intestine-specific LI-cadherin (Cdh17). Both genes consist of 18 exons and the positions of their exon-intron boundaries as well as their intron phases are perfectly conserved. We found an extensive paralogy of more than 40 megabases in mammals as well as teleost fish species encompassing the Ksp- and LI-cadherin genes. A comparable paralogy was not detected for other cadherin gene loci. These findings suggest that the Ksp- and LI-cadherin genes originated by chromosomal duplication early during vertebrate evolution and support our assumption that both proteins are paralogues within a separate cadherin family that we have termed 7D-cadherins.

Conserved co-regulation and promoter sharing of hoxb3a and hoxb4a in zebrafish

The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts, hoxb3a transcripts I–III and hoxb4a transcripts I–II, driven by four promoters. A ‘master’ promoter, located about 2 kb downstream of hoxb5a, controls transcription of a pre-mRNA comprising exon sequences of both genes. This unique gene structure is proposed to provide a novel mechanism to ensure overlapping, tissue-specific expression of both genes in the posterior hindbrain and spinal cord. Transgenic approaches were used to analyze the functions of zebrafish hoxb3a/ hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics. Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres (r) 5 to 8, suggesting a late role of the genes in neuronal cell lineage specification. Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6, whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5. We conclude that the similarity of hoxb3a/ Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene.

Polymorphism of UDP-Glucuronosyltransferase and Drug Metabolism

UDP-glucuronosyltransferase is a group of catabolic enzymes involved in the detoxification and excretion of many xenobiotic and endogeneous substances in intrahepatic and extrahepatic tissues. The group consists of two subfamilies, UGT1 and UGT2. UGT1 consists of 5 exons and has a unique gene structure. There are thirteen exon 1s from UGT1A1 to UGT1A13P, and exon 2 to exon 5 are used in common for all mRNAs expressed from the gene. Each isoform of UGT1 results from differential splicing of exon1s to common exon 2-5, and has an unique spectrum of substrate specificity. In contrast, the genes of the UGT2 family consist of 6 exons, and all the enzymes have an individual set of exon 1 to exon 6. In UGT1 there are no reports of polymorphism in the common exons, although a number of polymorphisms have been reported for exon 1s. The mutations of UGT1A1 cause hereditary unconjugated hyperbilirubinemias: Crigler-Najjar syndrome type I, type II and Gilbert syndrome. UGT1A1 has two major polymorphisms--a missense mutation of G71R and an insertion mutation of TATA box. Prevalence of Gilbert syndrome is attributed to these polymorphisms. Since UGT1A1 metabolizes not only bilirubin but also hormones and drugs, the mutations could be involved in carcinogenesis and adverse drug reactions. Recent studies also revealed a widespread presence of diverse polymorphisms in other isoforms of UGT1 as well as the UGT2 family, including UGT1A6, UGTG1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B7 and UGT2B15. The incidences and types of the polymorphisms for these enzymes are quite different in region and ethnic groups. Understanding of these polymorphisms is essential for the prevention of adverse effects of a considerable number of drugs and to predict cancer risks.

Conservation in divergent solanaceous species of the unique gene structure and enzyme activity of a gametophytically-expressed flavonol 3-O-galactosyltransferase.

Flavonol 3-O-galactosyltransferase (F3GalTase) is a pollen-specific enzyme which glycosylates the flavonols required for germination in petunia. The highly restricted tissue-specific expression and substrate usage make F3GalTase unique among all other flavonoid glycosyltransferases (GTs) described to date, including the well characterized Bronze 1 (Bz1) gene of maize. RFLP mapping, DNA gel blot, and sequence analyses showed that the single copy F3galtase gene has a different genomic organization than Bz1. Within the promoter of F3galtase are potential regulatory motifs which may confer pollen-specific gene expression and activation by Myb and bHLH transcription factors. However, we provide evidence that F3galtase is not regulated by An4, which encodes a Myb factor known to regulate anthocyanin accumulation in petunia anthers. An unexpected feature of the F3galtase promoter was the presence of large blocks of chloroplast and mitochondrial DNA. Gel blot analyses of genomic DNA from the progenitors of Petunia hybrida, as well as from Nicotiana tabacum, indicated that migration of organellar DNA into the F3galtase gene was an ancient event that occurred prior to speciation of the Solanaceae. Together with enzyme assays and HPLC analyses of pollen extracts from tobacco, tomato, and potato, these results confirmed that the unique F3galtase gene structure, enzyme activity, and pollen-specific flavonol glucosylgalactosides are conserved throughout the Solanaceae.

Both the conserved and the unique gene structure of stomach-specific calpains reveal processes of calpain gene evolution.

The proteins nCL-2 and nCL-2' are members of the Ca2+-dependent cysteine protease (calpain) superfamily, with stomach-specific expression. Like other typical calpains, nCL-2 has three distinct domains, a protease, a C2-like, and a 5EF-hand Ca2+-binding domain, as well as the N-terminal propeptide region. On the other hand, nCL-2' lacks the C2-like and 5EF-hand domains but is otherwise identical to nCL-2, except for the three C-terminal residues. To examine the stomach-specific and presumed alternative expression mechanisms of nCL-2 and nCL-2', we have cloned and characterized the mouse gene for nCL-2 and nCL-2'. The mouse nCL-2 gene contains at least 23 exons, spanning more than 50 kb, and possesses an exon specific for nCL-2' in the middle. Therefore, nCL-2 and nCL-2' are generated by alternative splicing of the same gene, Capn8. Capn8 shows the highly conserved gene organization of the other typical calpain large subunit genes, CAPN1, CAPN2, CAPN3, CAPN9, CAPN11, and Capn12, except for the unique exon between exon 9 and exon 10 of Capn8, which encodes the 3' half of the nCL-2' transcript. No such exon in the corresponding regions was found in CAPN1, CAPN2, CAPN3, CAPN9, or CAPN11. Gene and cDNA structures of a presumed human orthologue of mouse nCL-2, CAPN8, were determined, revealing that it overlaps human CAPN2, the gene for the m-calpain large subunit, in head-to-head orientation at 1q32-41. These features of Capn8 and CAPN8 illustrate a process of calpain gene evolution, i.e., the protease, C2-like, and 5EF-hand domains presumably functioned as independent genes, and the calpain superfamily has evolved by ordered fusions of these ancestral gene units, with subsequent amplifications.

Characterisation of a novel HLA-A pseudogene, HLA-BEL, with significant sequence identity with a gorilla MHC class I gene.

During the development of an HLA-A polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) method for the identification of HLA-A*24 and -A*30 alleles, group amplification resulted in the formation of an unusual PCR product in certain individuals. This fragment was approximately 900 bp smaller than the expected product and was also detected in some non-HLA-A*24- and -A*30-positive individuals acting as negative controls for the group specific amplification. Nucleotide sequence analysis of this product identified it as a unique class I gene sequence displaying homology to both primate and human class I A-locus genes. The entire gene was amplified using PCR and the complete DNA sequence information from exon 1 to exon 8, including introns, was determined. A recombination event was identified which results in the fusion of intron 2 with intron 3, causing a deletion of the intervening exon 3 sequence. In addition, there are two cytosine insertions in the poly-cytosine stretch at the start of exon 4 which cause a frameshift and premature termination. The exon 1 and 2 sequences most closely align with the gorilla allele A*0501, displaying only five mismatches. PCR analysis has established that the gene is associated with the following HLA-A types: HLA-A*3001, -A*3301, -A*3303, -A*6802, -A*2901, -A*0203, -A*0205 and -A*31012. Reverse transcription (RT)-PCR analysis of individuals containing this gene failed to detect any mRNA transcription, suggesting that this is a previously undescribed non-expressed class I pseudogene which we have provisionally named HLA-BEL. Its unique gene structure gives a possible insight into the evolutionary pathway that created HLA class I genes.

Serpins from an insect, Manduca sexta.

Members of the serpin superfamily have been identified in several invertebrate species, primarily as proteins in the hemolymph of animals from the phylum Arthropoda9. Serpin cDNAs have been cloned from horseshoe crabs14,15, a crayfish13, two species of lepidopteran insects6,10,17,20,24, the fruit fly Drosophila melanogaster 3, trematodes from the genus Schistosoma 2, and from a parasitic nematode25. The presence of serpins with differing proteinase inhibitory activities in the hemolymph of horseshoe crabs and insects suggests that serpins may function in several physiological processes in invertebrates, as they do in mammals. The number of invertebrate species vastly exceeds the number of vertebrates, with far greater biological diversity. Thus, it may be predicted that as biochemical studies on invertebrates progress, a large number of serpins with diverse activities and functions will be discovered. In our research on serpins from the tobacco hornworm, Manduca sexta, we have identified a family of serpins with different inhibitory activities, and a unique serpin gene structure for generating reactive site diversity.

Prolactin-like protein-C variant: complementary deoxyribonucleic acid, unique six exon gene structure, and trophoblast cell-specific expression

The rat placental PRL family consists of proteins structurally related to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we have termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy involving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence identity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of gestation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically activated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional activation.

17α-Hydroxylase/17,20-Lyase Deficiency: From Clinical Investigation to Molecular Definition*

THE term congenital adrenal hyperplasia (CAH) defines a group of syndromes resulting from inherited defects in any one of the five enzymes involved in the steps leading to cortisol biosynthesis from cholesterol. These include four distinct forms of cytochrome P450: P450scc, P450C21, P45011β, and P45017α, as well as 3β- hydroxysteroid dehydrogenase. Pathologically, CAH is characterized by hyperplastic growth of the adrenal stimulated by ACTH. A blockage in any one of the steps in normal steroid hormone synthesis leads to decreased synthesis of the steroid hormones and accumulation of intermediates, giving rise to the various clinical symptoms. The most common form of CAH, 21-hydroxylase deficiency, accounts for 90–95% of CAH (1). The high frequency of 21-hydroxylase deficiency is related to the structure of the P450C21 (CYP21) genes of which there are two, closely linked, a functional gene and a pseudo-gene (2, 3). Understanding of this unique gene structure and its location in the HLA locus has led to the...