Exon Organization Research Articles

Genomic Organization of Mouse and Human 65 kDa FK506-Binding Protein Genes and Evolution of the FKBP Multigene Family

FK506-binding proteins (FKBPs) are peptidyl–prolyl cis/trans isomerases PPIases) that bind the immunosuppressive drug FK506. Of the many eukaryotic FKBPs that have been identified, FKBP65 is an endoplasmic reticulum-localized protein that associates with tropoelastin in the secretory pathway. Unlike any other FKBP characterized so far, FKBP65 is developmentally regulated and may be intimately involved in organogenesis. Here, we report the isolation, sequencing, and genomic organization of the mouse FKBP65 gene (Fkbp10) and provide a comparison with the human ortholog. Mouse Fkbp10 contains 10 exons and 9 introns encompassing 8.5 kb. The exon–intron organization of Fkbp10 displays a pattern of repetition that reflects the coding sequence of the four PPIase, or FK506-binding, domains present in the mature protein. The exon organization of the PPIase domains differs from that of the other FKBP family members. The evolution of the FKBP65 gene and other members of the FKBP multigene family were therefore investigated from a taxonomically diverse array of prokaryotic and eukaryotic taxa. These analyses suggest that the FKBP multigene family emerged early in the evolutionary history of eukaryotes, and during that time some members, including the FKBP65 gene, have experienced gene elongation by means of PPIase domain duplication.

Identification of ten exon deleted ERβ mRNAs in human ovary, breast, uterus and bone tissues: alternate splicing pattern of estrogen receptor β mRNA is distinct from that of estrogen receptor α

Four different human tissues and breast cancer cell lines were screened to identify exon deletion variant transcripts of estrogen receptor β (ERβ) by reverse transcription-polymerase chain reaction using the ‘splice targeted primer approach’ that amplifies each category of exon deleted variants as a separate gene population. A total of 10 different variant mRNAs that have deletions in various combination of exons were identified by sequence analysis. They were exon 2Δ; exons 2 and 5–6Δ; exon 3Δ; exon 4Δ; exon 5Δ; exons 5 and 2Δ; exon 6Δ; exons 6 and 2Δ; exons 6, 2–3Δ; and exons 5–6Δ. In some cases, deletion of an exon appears to be associated with a mutation of a specific base. Although ERα and ERβ are highly homologous, have identical exon and functional domain organization, exhibit similar ligand-binding profiles and interact with identical DNA response elements, the sequence of exon skipping in ERβ pre-mRNA appears to be distinct from that of ERα mRNA. Furthermore, results described here also suggest that alternate splicing of ERβ mRNA is tissue specific. The presence of a ERβ variant profile together with other ER isoforms in a tissue may have functional implications in binding and response to a particular ligand.

A Conserved Sequence within the Propeptide Domain of Membrane Type 1 Matrix Metalloproteinase Is Critical for Function as an Intramolecular Chaperone

The propeptide domain of secreted matrix metalloproteinases (MMPs) is responsible for maintaining the latency of these proteinases. Recently, the propeptide domain of the prototype membrane type matrix metalloproteinase (MT1-MMP) was demonstrated to act as an intramolecular chaperone (Cao, J., Hymowitz, M., Conner, C., Bahou, W. F., and Zucker, S. (2000) J. Biol. Chem. 275, 29648-29653). In the current study, the role of an unique four-amino acid sequence in the propeptide domain of MT1-MMP was examined. The sequence (42)YGYL(45) is conserved in the propeptide domain of all six members of the MT-MMP subfamily, but not in secreted MMPs. Mutant MT1-MMP cDNAs coding for alanine substitutions (single and double amino acid sequences) in this conserved propeptide region were transfected into COS-1 cells deficient in endogenous MT1-MMP. As demonstrated by immunofluorescence, mutant MT1-MMP protein was synthesized and displayed on the plasma membrane of transfected cells. Alanine substitutions within the (42)YGYL(45) sequence proved to be detrimental for enzyme function in terms of activation of proMMP-2 and binding TIMP-2 to the cell surface (MT1-MMP serves as a cell surface receptor for TIMP-2). In contrast to wild-type MT1-MMP-transfected cells, mutant MT1-MMP-transfected cells were incapable of degrading and migrating on a fibronectin substrate. These data indicate that the conserved (42)YGYL(45) sequence within the propeptide domain of MT-MMPs is required for intramolecular chaperone function of these intrinsic membrane proteinases.

The novel human HUEL (C4orf1) protein shares homology with the DNA-binding domain of the XPA DNA repair protein and displays nuclear translocation in a cell cycle-dependent manner

We have previously isolated and characterized a novel human gene HUEL ( C4orf1) that is ubiquitously expressed in a wide range of human fetal, adult tissues and cancer cell lines. HUEL maps to region 4p12-p13 within the short arm of chromosome 4 whose deletion is frequently associated with bladder and other carcinomas. Here we present the genomic organization, sizes and boundaries of exons and introns of HUEL. The GC-rich upstream genomic region and 5′ untranslated region (UTR) together constitute a CpG island, a hallmark of housekeeping genes. The 3250 bp HUEL cDNA incorporates a 1704 bp ORF that translates into a hydrophilic protein of 568-amino acids (aa), detected as a band of ∼70 kDa by Western blotting. We have isolated the murine homolog of HUEL which exhibits 89% nucleotide and 94% amino acid identity to its human counterpart. The HUEL protein shares significant homology with the minimal DNA-binding domain (DNA-BD) of the DNA repair protein encoded by the xeroderma pigmentosum group A ( XPA) gene. Other notable features within HUEL include the putative nuclear receptor interaction motif, nuclear localization and export signals, zinc finger, leucine zipper and acidic domains. Mimosine-mediated cell cycle synchronization of PLC/PRF/5 liver cancer cells clearly portrayed translocation of HUEL into the nucleus specifically during the S phase of the cell cycle. Yeast two-hybrid experiments revealed interactions of HUEL with two partner proteins (designated HIPC and HIPB) bearing similarity to a mitotically phosphorylated protein and to reverse transcriptase. Co-immunoprecipitation assays validated the interaction between HUEL and HIPC proteins in mammalian cells. HUEL is likely to be an evolutionarily conserved, housekeeping gene that plays a role intimately linked with cellular replication, DNA synthesis and/or transcriptional regulation.

A comparative analysis of invaded sequences from group IA phospholipase A2 genes provides evidence about the divergence period of genes groups and snake families

Two phospholipase A2 (PLA2) genes classified into group IA were cloned from the genomic library of the sea snake Laticauda semifasciata. Eight clones were obtained by PCR cloning procedure from genomic DNA of Laticauda laticaudata (four clones) and Laticauda colubrina (four clones). The genes were 3.6–4.4kbp in length. Intron and exon organization of the group IA PLA2 genes was the same as that of Naja sputatrix group IA PLA2 genes (four exons and three introns). There were two kinds of repetitive sequences in the first and second introns of all sequenced PLA2 genes. The differences in the length of these genes were derived from the length of their repetitive sequences. The chicken repeat-1 (CR1)-like long interspersed repeated DNA (LINE) sequences, different from the above repetitive sequences, were also found in all sequenced Laticauda PLA2 genes. A comparative analysis of groups IA, IA′ and IIA PLA2s genes suggests a period of CR1-like LINE integration during molecular and family evolution. The integration of CR1-like LINE into PLA2 genes occurred after the divergence of groups I and II PLA2s but before the divergence of groups, IA and IA′ PLA2s. These integration events occurred before the family divergence of Naja and Laticauda. The presence of CR1-like LINE and a comparison of intron and exon organization showed that the divergence of Naja and Bungarus occurred before the divergence of Laticauda and Naja.

ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis.

The Na+-dependent amino acid transporter named ATB(0) was previously found to be located in 19q13.3 by fluorescence in situ hybridisation. Genetic heterogeneity in the 19q13.2-13.4 region, syntenic to the Cystic Fibrosis Modulator Locus 1 (CFM1) in mouse, seemed to be associated to the intestinal phenotypic variation of cystic fibrosis (CF). We performed fine chromosomal mapping of ATB(0) on radiation hybrid (RH) panels G3 and TNG. Based on the most accurate location results from TNG-RH panel, mapping analysis evidenced that ATB(0) is localised between STS SHGC-13875 (D19S995) and STS SHGC-6138 in 19q13.3, that corresponds with the immediately telomeric/distal segment of the strongest linkage region within the human CFM1 (hCFM1) syntenic region. Regarding to the genomic structure and exon organisation, our results show that the ATB(0) gene is organised into eight exons. The knowledge of the genomic structure allowed us to perform an exhaustive mutational analysis of the gene. Evaluation of the possible implication of ATB(0) in the intestinal phenotype of CF was performed on the basis of the functional characteristics of the encoded protein, its apparent relevance to meconium ileus (MI) and position in relation to the hCFM1 syntenic region. We have analysed this gene in samples from CF patients with and without MI. Several sequence variations in the ATB(0) gene were identified, although none of them seemed to be related to the intestinal phenotype of CF. Even though no particular allele or haplotype in ATB(0) appears to be associated to CF-MI disease, new SNPs identified should be useful in segregation and linkage disequilibrium analyses in families affected by other disorders caused by the impairment of neutral amino acid transport.

Gene cloning of a new plasma CC chemokine, activating and attracting myeloid cells in synergy with other chemoattractants.

Chemokines are important mediators of cell migration during inflammation and normal leukocyte trafficking. Inflammatory chemokines are induced in multiple cell types at sites of infection. Here, we describe a novel bovine CC chemokine, designated regakine-1, that is constitutively present at high concentrations in plasma. Cloning of its gene revealed an expected two intron/three exon organization, with a rather long first intron. In addition to a 21-residue signal peptide, the coding sequence corresponded to a 71-residue secreted protein. However, the natural regakine-1 protein missed the COOH-terminal lysine residue. Regakine-1 has only weak sequence similarity (<50% identical residues) with other animal or human chemokines. Northern blot analysis demonstrated regakine-1 RNA expression in spleen and lung. At physiological concentrations (30-100 ng/mL), natural 7.5 kDa regakine-1 stimulated gelatinase B release from neutrophils and chemoattracted immature myeloid HL-60 cells, as well as mature granulocytes. Regakine-1 was more potent on human myeloid cells than the human plasma CC chemokine hemofiltrate CC chemokine-1 (HCC-1). Moreover, regakine-1 synergized with the bacterial peptide N-formylmethionylleucylphenylalanine (fMLP), yielding a 10-fold increase in neutrophil chemotactic response above their additive effect. Regakine-1 did not compete with interleukin-8 (IL-8) for binding to neutrophils, nor did it affect fMLP-induced calcium signaling, suggesting that regakine-1 recognizes a different receptor. In view of its high constitutive plasma concentration, regakine-1 is believed to recruit myeloid cells into the circulation, whereas its synergy with other neutrophil chemoattractants suggests that it also enhances the inflammatory response to infection.

Structural organization of the rat mitogen-activated protein kinase phosphatase 2 gene

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are dual specificity protein phosphatases that specifically inactivate MAPKs. Regulated expression of MKPs plays a key role in determining their physiological function. However, little is known about the molecular mechanism of the activation of MKP genes. In this study, we cloned the rat MKP-2 gene and characterized its structure. The MKP-2 gene has four exons and three introns. The organization of exons of the MKP-2 gene is very similar to that of the MKP-1 gene, suggesting that MKP-1 and MKP-2 are derived from the same ancestral gene. We identified multiple transcription start sites (TSSs) for the MKP-2 gene. There is no functional TATA motif in the 5′ proximal region of the TSSs. Instead, this region is highly GC-rich and has two putative Sp1 sites. A 1.8 kb 5′ flanking region of the MKP-2 gene is sufficient to mediate transcriptional activation of the luciferase reporter gene by phorbol ester in GH3 cells. These results provide essential information about structural organization and regulatory sequences of the MKP-2 gene for further investigation of the molecular mechanisms of MKP-2 induction by extracellular stimuli.

Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cladocera, Moina macrocopa, were cloned and their nucleotide (nt) sequences were determined. The amino acid (aa) sequences of both the gene products deduced from the nt sequences consisted of 348 residues and showed 98% identity with each other. These sequences together with the NH 2-terminal aa sequences of the Hb chains determined after separation by two-dimensional gel electrophoresis showed that the Hb chains are synthesized as a secretory precursor with a signal peptide of 17 aa residues. The aa sequences of M. macrocopa Hb chains shared the following features with those of Daphnia Hb chains. Firstly, the signal peptide is followed by an NH 2-terminal extension containing a threonine-rich sequence that might play a role in the multimerization of subunit chains. Secondly, the identity between the aa sequences of the first and second domains is exceptionally low. These facts suggest that duplication of the cladoceran Hb gene occurred before the divergence of families Moinidae and Daphniidae. Analysis of genomic DNA showed that the M. macrocopa Hb genes consist of two large repeated regions, encoding the first and second domains of Hb chains, respectively. The intron–exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure characteristic of animal Hb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moina genes, providing a new example of intron loss. The following elements in the 5′ flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, GAAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moina); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sites for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia.

Use of an in Silico Approach to Define the Gene Structure of Eukaryotic Adenylyl Cyclases

The limited information available regarding the gene structure of adenylyl cyclases (AC), which catalyze the synthesis of cAMP, suggests a complex arrangement with many exons and large introns such that molecular techniques to define these gene structures are time- and labor-intensive. We report here the use of a computer-based approach involving the assembly of fragmented sequence data generated by the Human Genome Project and nucleic acid analysis software to decipher the gene structure of human and murine AC 6 and other human AC isoforms (ACs 3, 7, and 8). The results, which document 21 exons in human and murine AC 6, human AC 3, 18 exons in AC 8, and 24 exons in AC 7, show substantial conservation of exon organization in the AC family and in particular regions of the AC protein. Application of such in silico methods should prove useful to characterize genes for other ACs and protein families and data provided here should facilitate studies of polymorphisms in AC genes.

Genetic and functional analysis of PARP, a DNA strand break-binding enzyme

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by binding to a single- or double-strand break of DNA and is one of the death substrates for caspase-3 in apoptosis. The nuclear function of PARP is well studied and recent PARP-knockout studies indicate that PARP takes part in chromosomal stability. To analyze the effect of PARP overexpression, or loss of function, we have cloned PARP cDNA and the gene from Drosophila melanogaster and studied its function in developmental stages. Organization of exons corresponds to the functional domains of PARP. An alternatively spliced form of PARP lacking exon 5, which encodes the auto-modification domain, is found in Drosophila. Expression of the PARP gene is at high levels in embryos at 0–6 h after egg laying and gradually decreased. In situ mRNA hybridization indicates localization of PARP mRNA in cells along the central nervous system at a late stage of embryogenesis. Overexpression of the gene in the developing eye primordia of D. melanogaster is an excellent experimental model to analyze the cell cycle and programmed cell death. We introduced PARP expression vector overexpresses PARP in the eye discs of Drosophila, and established the PARP transgenic flies by P element-mediated germ line transformation. These flies showed mild roughening of the normally smooth ommatidial lattice involving tissue polarity disruption characterized by missrotation and incorrect chirality of ommatidia. Possible mechanisms of involvement of PARP in the development are discussed.

Conserved Structure and Promoter Sequence Similarity in the Mouse and Human Genes Encoding the Zinc Finger Factor BERF-1/BFCOL1/ZBP-89

We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5′-end region, 9 exons, 8 introns, and the 3′-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allowed us to determine that the same genomic structure with identical intron–exon organization is conserved in the human homologue ZNF 148. Functional analysis of the 5′-flanking sequence of the mouse gene indicated that the region from nucleotide −205 to +144, relative to the major transcription start site, contains cis-regulatory elements that promote basal expression. Such sequences and the overall promoter architecture are highly conserved in the human gene. Furthermore, we show that the complex transcription pattern of the Zfp148 gene might be due to a combination of alternative splicing and differential polyadenylation sites utilization.

A genomic clone for a chitinase gene from the silkworm, Bombyx mori: structural organization identifies functional motifs

A genomic clone containing the chitinase gene was isolated and characterized from the silkworm, Bombyx mori, using the polymerase chain reaction technique directed by primers designed from the chitinase cDNA of the insect previously cloned [Insect Biochem. Molec. Biol. 28 (1998) 163]. Results from nucleotide sequence analysis of the gene and its PCR amplification suggest that the B. mori genome probably has only one copy of chitinase gene. The gene is organized into 10 coding regions, exons, interrupted by noncoding regions, introns. The motifs encoded and exon organization of the gene are almost identical to the related gene from Manduca sexta. The results suggest that the domain organization of chitinase genes may be conserved among insect cuticular chitinase genes and that they are more complex than their counterparts in plants. The possibilities of intron splicing from the primary transcripts are also discussed.

Genomic Organization, Amplification, Fine Mapping, and Intragenic Polymorphisms of the Human Hemidesmosomal Tetraspanin CD151 Gene

CD151 is a member of the tetraspanin family that is involved in cellular processes including cell adhesion. The protein is expressed in a variety of tissues including vascular endothelium and epidermis, and has been shown to be a component of hemidesmosomes. Mutations in genes encoding other hemidesmosomal proteins give rise to a range of human disorders, characterized by fragility of the skin and/or mucous membranes. It is, therefore, plausible that inherited or acquired mutations in the gene encoding CD151 may be fundamental to the integrity and maturation of basal cell keratinocytes. To aid mutation analysis, we have characterized the intron–exon organization of the CD151 gene which comprises 8 exons spanning approximately 4.3 kb, and have developed a comprehensive PCR-based mutation detection strategy. In addition, to aid linkage analysis of CD151 in genetic disease we have fine-mapped the gene by radiation-hybrid methodology to 11p15.5, and detected a number of intragenic polymorphisms.

Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (&lt; 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.

Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements

AbstractTo understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.

Structural characterization and chromosomal localization of the mouse cDNA and gene encoding the bone tyrosine phosphatase, mOST-PTP

Tyrosine kinases and phosphatases are regulators of the steady-state levels of phosphotyrosine proteins and, in this way, are key players in determining the functional state of the cell. As a unique member of the protein tyrosine phosphatase (PTP) superfamily, osteotesticular PTP (OST-PTP) is a receptor protein whose expression is highly regulated during osteoblast differentiation and in response to modulators of bone remodeling such as parathyroid hormone and vitamin D3. To explore the molecular mechanisms and signaling pathways important in the regulation of this gene, we characterized the structural organization of the mouse OST-PTP cDNA and gene and determined its chromosomal localization. The mouse cDNA is ∼5.5 kb including 5.1 kb of coding sequence, 315 bp 5′ UTR and 102 bp 3′ UTR. It is expressed as a single ∼5.8 kb transcript in day 8 differentiated MC3T3 osteoblasts. Although highly homologous to the rat OST-PTP cDNA, the mouse cDNA possesses a 74 bp insert in the 5′ UTR which contains several potential transcription factor binding sites such as AP-2 and NFκB. The mouse OST-PTP (mOST-PTP) gene is a single copy gene encompassing 35 exons and spanning only 20.65 kb. As such, it is the smallest gene of the characterized receptor PTP genes. This is due to the lack of large introns and the conserved spatial organization of exons which encode specific protein motifs in the mOST-PTP molecule. Sequence analysis of the putative mOST-PTP promoter revealed basal elements as well as many potential cis-acting regulatory elements with relevance to gene regulation in bone. Of particular interest is the single osteoblast specific element known as osteocalcin specific element 2 (OSE2) found at position −1867, as well as numerous VDRE and NFκB sites found throughout the promoter and the 5′ UTR. Fluorescence in situ hybridization studies have shown that mOST-PTP localizes to mouse chromosome 1, region F–G which is syntenic to the segment of human chromosome 1q32–33. This characterization of the mOST-PTP cDNA and gene will facilitate future experiments exploring the mechanisms of regulation of this phosphatase during osteogenesis.

A cytochrome P4501B gene from a fish, Pleuronectes platessa

Tetrapod cytochrome P4501 family (CYP1A1, CYP1A2 and CYP1B1) enzymes are most active in hydroxylating a variety of environmental contaminants including polyaromatic hydrocarbons (PAH), planar polychlorinated biphenyls and arylamines and thus play a pivotal role in the toxicology of these compounds. Mammalian CYP1A1 and CYP1A2 genes appear to have diverged after the evolutionary emergence of mammals, whereas fish species apparently possess only one CYP1A family gene, and fish CYP1A enzymes exhibit properties of both of the mammalian isoforms. We have isolated a further CYP1 family gene from a marine flatfish (plaice; Pleuronectes platessa), which, on the basis of exon organisation and sequence similarity, can be assigned as a piscine CYP1B. Its deduced amino acid sequence shows the closest (54%) identity to mammalian CYP1B1 proteins and, on the basis of molecular modeling studies, shows a high degree of positional and structural conservation of the substrate contacting amino acid residues in its putative active site when compared to other CYP1 enzymes. Phylogenetic analysis of fish and mammalian CYP1 family sequences indicates that the plaice CYP1B and mammalian CYP1B1 genes share a common ancestry. Plaice CYP1B has a more restricted tissue expression profile than the previously isolated plaice CYP1A, only being detectable, by Northern blotting, in gill tissue. In contrast to CYP1A, which shows extensive PAH-dependent induction in a variety of tissues, plaice CYP1B appears unresponsive to treatment with a prototypical PAH-type inducer, β-naphthoflavone (BNF).

The human phosphodiesterase PDE10A gene

PDE10A is a cyclic nucleotide phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. The transcripts are specifically expressed in the striatum. The human gene encoding PDE10A was cloned and investigated. The PDE10A gene spanned > 200 kb and contained 24 exons. The exon-intron organization of PDE10A was different from those of PDE5A and PDE6B, although these three PDEs include two GAF domains and have similar amino-acid sequences. The promoter sequence of PDE10A was highly GC-rich and did not contain a TATA motif and a CAAT box, suggesting it is a housekeeping gene. In Caenorhabditis elegans, the C32E12.2 gene encoding a probable PDE that is 48% identical to the human PDE10A protein showed similar exon organization to PDE10A but not PDE5A and PDE6B. This, together with the phylogenic tree analysis, suggested that the ancestral gene for PDE10A existed in a lower organism such as C. elegans.

Genomic organization of mouse and human erythrocyte tropomodulin genes encoding the pointed end capping protein for the actin filaments

Erythrocyte tropomodulin (E-Tmod), a globular protein of 359 residues, is highly expressed in the erythrocyte, heart and skeletal muscle. By binding to the N-terminus of tropomyosin (TM) and actin, E-Tmod blocks the elongation and depolymerization of the actin filaments at the pointed end. In erythrocytes, the E-Tmod/TM complex contributes to the formation of the short actin protofilament, which in turn defines the geometry of the membrane skeleton. In juvenile mice, over-expression of E-Tmod is associated with dilated cardiomyopathy. We have previously cloned the human E-Tmod cDNA, identified its TM-binding region, and mapped its gene to chromosome 9q22. Through genomic library screening and PCR-based genomic walking we have now cloned the mouse E-Tmod gene, whose coding region spans ∼60 kb containing nine exons and eight introns. The human E-Tmod gene obtained by PCR has an identical exon–intron organization. In sanpodo, a Tmod homologue in Drosophila, the exon boundaries are also conserved except that exons 2–5 and 6–7 are ‘fused’ and alternative splicing of two additional 5′ exons and the 3′ exons may give rise to several sanpodo isoforms. In a Tmod-like gene of C. elegans, exons 2–3 are ‘fused’, boundaries of exons 1, 7, 8, and 9 are conserved and exon/intron junctions of exons 4, 5 and 6 are shifted by a few residues. Analyses of 15 Tmod members from six species show no insertions or deletions of residues in the region of exons 6 and 7. A 5′ rapid amplification of cDNA ends reveals that mouse E-Tmod transcripts obtained from embryonic stem cells, skeletal muscle and heart, but not smooth muscle, contain an additional 86 bp untranslated cDNA sequence further upstream from exon 1. Thus, alternative promoters may provide a possible mechanism for tissue-specific expression and regulation of E-Tmod. This study is the first to report the exon organization of E-Tmod genes, which allows their regulation, manipulation, and disease relevance to be further investigated.