A novel zebrafish kelchlike gene klhl and its human ortholog KLHL display conserved expression patterns in skeletal and cardiac muscles.

In this study, a novel gene, kelchlike (klhl) was identified in zebrafish by whole-mount in situ hybridization screen for important genes involved in embryogenesis. A full-length klhl cDNA was cloned and characterized. We found that klhl was a member of the kelch-repeat superfamily, containing two evolutionary conserved domains--broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, and kelch motif. Database mining revealed the presence of putative orthologs of klhl in human, mouse, rat, and pufferfish. klhl was determined to map to zebrafish linkage group (LG) 13 and was found to be syntenic with the proposed orthologs of klhl in human, mouse, and rat. In an effort to elucidate the function of klhl, klhl expression was investigated by Northern blot analysis and in situ hybridization. klhl is specifically expressed in the fast skeletal and cardiac muscle. Northern blot analyses show that the human ortholog, KLHL, is also specifically expressed in the skeletal muscles and heart. In silico analyses of rat expressed sequence tag (EST) clones corresponding to rat Klhl ortholog also indicate that its expression is also restricted to rat muscle tissues, suggesting a conserved role of klhl in vertebrates. The expression pattern of klhl, as well as the presence of the kelch repeats indicates a possible role for Klhl in the organization of striated muscle cytoarchitecture.

Wide-cross whole-genome radiation hybrid mapping of cotton (Gossypium hirsutum L.).

We report the development and characterization of a "wide-cross whole-genome radiation hybrid" (WWRH) panel from cotton (Gossypium hirsutum L.). Chromosomes were segmented by gamma-irradiation of G. hirsutum (n = 26) pollen, and segmented chromosomes were rescued after in vivo fertilization of G. barbadense egg cells (n = 26). A 5-krad gamma-ray WWRH mapping panel (N = 93) was constructed and genotyped at 102 SSR loci. SSR marker retention frequencies were higher than those for animal systems and marker retention patterns were informative. Using the program RHMAP, 52 of 102 SSR markers were mapped into 16 syntenic groups. Linkage group 9 (LG 9) SSR markers BNL0625 and BNL2805 had been colocalized by linkage analysis, but their order was resolved by differential retention among WWRH plants. Two linkage groups, LG 13 and LG 9, were combined into one syntenic group, and the chromosome 1 linkage group marker BNL4053 was reassigned to chromosome 9. Analyses of cytogenetic stocks supported synteny of LG 9 and LG 13 and localized them to the short arm of chromosome 17. They also supported reassignment of marker BNL4053 to the long arm of chromosome 9. A WWRH map of the syntenic group composed of linkage groups 9 and 13 was constructed by maximum-likelihood analysis under the general retention model. The results demonstrate not only the feasibility of WWRH panel construction and mapping, but also complementarity to traditional linkage mapping and cytogenetic methods.

Spatio-temporal distribution of cellular retinol-binding protein gene transcripts (CRBPI and CRBPII) in the developing and adult zebrafish (Danio rerio).

We have cloned and determined the nucleotide sequence of the cDNA coding for a cellular retinol-binding protein type I (CRBPI) from zebrafish. The deduced amino acid sequence of the zebrafish CRBPI showed highest sequence identity ( approximately 59%) to the mammalian CRBPIs of the intracellular lipid-binding protein (iLBP) multigene family. Phylogenetic analysis clustered the zebrafish CRBPI to the CRBPI clade. The zebrafish CRBPI gene (rbp1) and CRBPII gene (rbp2) both consist of four exons separated by three introns, identical to all other iLBP genes in vertebrates. Two transcription start sites were identified in the rbp1 promoter and a single transcription start site was identified for rbp2. Radiation hybrid mapping assigned the zebrafish rbp1 gene to linkage group 16 and conserved syntenic genes were found by comparative analysis of mammalian orthologous rbp1 genes. RT-PCR detected mRNA transcripts in the adult intestine, liver, brain, ovary and testis for rbp1 gene and in the intestine and liver for rbp2 gene. Whole mount in situ hybridization of zebrafish embryos revealed rbp1 mRNA expression in the developing zebrafish central nervous system at specific sites that are known to have abundant retinoic acid distribution and significant retinoic acid action. Whole mount in situ hybridization also showed that the zebrafish rbp2 mRNA was localized specifically in the embryonic intestinal bulb and the developing intestine during the larval stage, implying a novel function for the rbp2 gene product during organogenesis and development of the zebrafish intestine.

Skin-specific expression of ictacalcin, a homolog of the S100 genes, during zebrafish embryogenesis.

Full-length cDNA coding for the ictacalcin gene, a homolog of the S100 genes, was isolated in zebrafish and mapped on linkage group 16 using the LN54 radiation hybrid panel. The homology and phylogenetic analyses, based on the deduced amino acid sequences, showed the orthologous relationship of ictacalcin genes between zebrafish and other fish species. However, ictacalcin genes constitute an out-group with respect to other members of the S100 gene family. This result supports the findings that fish ictacalcin genes are new members of the S100 gene family and may have evolved after the divergence of teleosts and tetrapods. The zebrafish ictacalcin gene was zygotically transcribed from 12 hours postfertilization onward and was stably expressed throughout adulthood. During zebrafish embryogenesis, the ictacalcin gene was specifically expressed in striated epidermal cells covering the entire embryo. The ictacalcin staining in keratinocytes of striated epithelia was absent in the cytoplasm surrounding the nuclei, but it was highly concentrated in the peripheral margin. Tissues enriched with epithelia folds, such as olfactory epithelium, hatching gland, pectoral fin buds, urogenital opening, and pharynx, showed a robust ictacalcin expression. The strikingly heavy staining of ictacalcin in the pharyngeal region provides us with an early marker to follow the pharynx formation in zebrafish embryos.

Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2).

We report the isolation and characterization of human contactin 4 (CNTN4), a brain-derived, immunoglobulin superfamily molecule-2 (alias BIG-2) as a candidate gene responsible for the differentiation potential of human neuroblastoma cells. Northern blot analysis showed highest CNTN4 expression in testes, thyroid, small intestine, uterus and brain. Induction of CNTN4 mRNA expression in human neuroblastoma tumor cells treated with retinoic acid correlated with a block in retinoid-induced neuritogenesis. Our findings suggest a role for human contactin 4 protein in the response of neuroblastoma cells to differentiating agents.

A cellular retinoic acid-binding protein from zebrafish (Danio rerio): cDNA sequence, phylogenetic analysis, mRNA expression, and gene linkage mapping.

We report the sequence of a cDNA clone coding for a cellular retinoic acid-binding protein (CRABP) in zebrafish. The encoded polypeptide is 142 amino acids in length with an estimated molecular mass of 15.8 kDa and a calculated isoelectric point of 5.2. The zebrafish CRABP exhibits highest sequence identity to the pufferfish CRABPIIa (83%) and CRABPIIb (79%), and human CRABPII (74%) than to any other member of the intracellular lipid-binding protein (ILBP) family. A phylogenetic tree for different members of the ILBP multigene family including fatty acid-binding proteins (FABPs), cellular retinol-binding proteins (CRBPs) and CRABPs shows that the cloned zebrafish cDNA encodes a protein that clusters with CRABPs from other species and not with CRBPs and FABPs. Reverse-transcription polymerase chain reactions (RT-PCR), using oligonucleotide primers specific to the zebrafish CRABP cDNA made from total RNA of embryos collected at various developmental stages, did not detect the CRABP mRNA until 12 h post-fertilization. In adult zebrafish, CRABP mRNA was detected by RT-PCR in total RNA extracted from muscle, testes and skin, barely detectable in heart, ovary and brain and undetectable in liver, kidney and intestine. Quantitative RT-PCR (qRT-PCR) revealed a similar tissue-specific distribution for zebrafish CRABP mRNA with highest levels of CRABP mRNA in muscle followed by testes, skin, ovary and much lower levels in heart. Radiation hybrid mapping assigned the CRABP gene to linkage group 16 in the zebrafish genome. Comparison of the mapped zebrafish CRABP and human CRABPII genes revealed that zebrafish linkage group 16 has a syntenic relationship with human chromosome 1. Based on phylogenetic analysis and the syntenic relationship to the CRABPII gene in human, the zebrafish cDNA clone appears to code for a type II CRABP.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. METHODS: A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. RESULTS: Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. CONCLUSIONS: The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Molecular cloning, mapping and characterization of a novel mouse RING finger gene, Mrf1.

With a combined approach of database search, heterologous polymerase chain reaction (PCR), reverse transcription-PCR, rapid amplification of complementary DNA ends and genomic library screening, we have successfully cloned a mouse RING finger gene, mouse RING finger 1 (Mrf1). The Mrf1 gene has two exons of 63 and 2665 bp, respectively, and one intron of over 13 kb. An open reading frame was identified exclusively in exon 2, which encodes a putative protein of the RING-B box-coiled coil or the tripartite motif type of 403 amino acids. Mrf1 is moderately expressed in the spleen, brain and heart as a single 3.0 kb product and very highly expressed in the testis as two transcripts of 3.0 and 1.5 kb, respectively. The Mrf1 gene was mapped to mouse chromosome 3, between markers D3Mit70 and D3Mit277. Western blotting analysis indicated that an expected protein of approximately 44 kD was detected in the brain extracts of mouse, rat and human. The possible functions of Mrf1 are discussed in the contexts of protein-protein interactions, oncogenesis and ubiquitination.

Cysteine and tyrosine-rich 1 (CYYR1), a novel unpredicted gene on human chromosome 21 (21q21.2), encodes a cysteine and tyrosine-rich protein and defines a new family of highly conserved vertebrate-specific genes.

A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis. Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein. The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1). The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb). The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb. Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system. We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21. Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins. The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.

BFIT, a unique acyl-CoA thioesterase induced in thermogenic brown adipose tissue: cloning, organization of the human gene and assessment of a potential link to obesity.

We hypothesized that certain proteins encoded by temperature-responsive genes in brown adipose tissue (BAT) contribute to the remarkable metabolic shifts observed in this tissue, thus prompting a differential mRNA expression analysis to identify candidates involved in this process in mouse BAT. An mRNA species corresponding to a novel partial-length gene was found to be induced 2-3-fold above the control following cold exposure (4 degrees C), and repressed approximately 70% by warm acclimation (33 degrees C, 3 weeks) compared with controls (22 degrees C). The gene displayed robust BAT expression (i.e. approximately 7-100-fold higher than other tissues in controls). The full-length murine gene encodes a 594 amino acid ( approximately 67 kDa) open reading frame with significant homology to the human hypothetical acyl-CoA thioesterase KIAA0707. Based on cold-inducibility of the gene and the presence of two acyl-CoA thioesterase domains, we termed the protein brown-fat-inducible thioesterase (BFIT). Subsequent analyses and cloning efforts revealed the presence of a novel splice variant in humans (termed hBFIT2), encoding the orthologue to the murine BAT gene. BFIT was mapped to syntenic regions of chromosomes 1 (human) and 4 (mouse) associated with body fatness and diet-induced obesity, potentially linking a deficit of BFIT activity with exacerbation of these traits. Consistent with this notion, BFIT mRNA was significantly higher ( approximately 1.6-2-fold) in the BAT of obesity-resistant compared with obesity-prone mice fed a high-fat diet, and was 2.5-fold higher in controls compared with ob/ob mice. Its strong, cold-inducible BAT expression in mice suggests that BFIT supports the transition of this tissue towards increased metabolic activity, probably through alteration of intracellular fatty acyl-CoA concentration.

Cloning of a novel retinoid-inducible serine carboxypeptidase from vascular smooth muscle cells.

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an amino-terminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates an approximately 51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein. Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.

We have identified a novel human gene, designated C1orf10, using modified differential display PCR. The C1orf10 gene, which spans 5 kb in length, is composed of three exons. The deduced protein contains 495 amino acids with one transmembrane domain. The amino acid sequence of C1orf10 is characterized by the presence of a calcium-binding motif of about 90 amino acids at its N-terminal and a conserved consecutive repeat sequence of 60 amino acids that was identified previously only in bacterial ice nucleation proteins. In normal adult tissues, C1orf10 is highly expressed only in the esophagus and was undetectable in a total of 15 other tissues examined, suggesting its important role in esophageal cells. The expression of C1orf10 is either dramatically reduced or absent in esophageal cancer cell lines (3/3) as well as primary esophageal cancer tissues (35/37) compared with the corresponding normal esophageal mucosa. Using a radiation hybrid panel, C1orf10 was found to be located on chromosome 1q21. These findings suggest that expression of C1orf10 is unique to esophageal cells and that loss of its expression may play a role in the development of esophageal cancer.

Characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1) mapping to 22q13.1.

This paper reports the characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1), which maps to the chromosome region 22q13.1 and has been partially duplicated on three other acrocentric chromosomes: 13, 15 and 21. We describe the complete cDNA, TTLL1a, coding for the putative 423 amino acid long TTLL1 and alternative transcripts coding for truncated TTLL1. Likely TTLL1a corresponds to the 1.8 kb transcript that was detected in a wide range of tissues and has a stronger expression in heart, brain and testis. A 4.8 kb transcript was found only in brain tissues. We present an interspecies sequence comparison, revealing three conserved domains, named TTLD1, TTLD2 and TTLD3, that are specific to the TTLs and TTL-like proteins.

The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.

The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.