Isolation of a ubiquitin-like (UBL5) gene from a screen identifying highly expressed and conserved iris genes

We have screened a human adult iris cDNA library to identify genes that are highly expressed and conserved between humans and pigs. We identified human iris cDNAs that hybridized at high stringency to a porcine choroidal ring cDNA probe. Of 1568 human iris cDNAs examined, 176 were found to have high expression in porcine choroidal rings. One of the 176 clones was identified as a previously uncharacterized cDNA that we have named the Ubiquitin-like 5 gene (UBL5). The UBL5 gene is located on chromosome 19p13.2, and its genomic structure has been examined. There is a UBL5 pseudogene on chromosome 17p11.2. We have also found homologues to the UBL5 gene in Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Northern blot analysis of the Ubiquitin-like gene 5 revealed expression in every tissue tested, with the highest levels of RNA expression in heart, skeletal muscle, kidney, liver, iris, and lymphoblasts. Intracellular localization experiments in COS-7 cells showed that the recombinant UBL5 protein is cytoplasmic. Western analysis demonstrated that the recombinant UBL5 protein is approximately 9 kDa, as predicted from the cDNA. A comparison between UBL5 and its homologues with other Ubiquitin-like proteins and Ubiquitin, using the PROTDIST program, suggests that the UBL5 genes are a separate class of Ubiquitin-like genes. Further characterization of the UBL5 gene will determine the function of the encoded protein and whether it is a candidate for ocular disease.     

Identification,genomic organization and mRNA expression of CRELD1, the founding member of a unique family of matricellular proteins

We have isolated and characterized a unique gene that encodes a highly conserved membrane bound extracellular protein that defines a new epidermal growth factor-related gene family. The CRELD1 (Cysteine-Rich with EGF-Like Domains 1) gene (previously known as cirrin) was cloned from a human chromosome 3 BAC. Mapping of the gene confirmed its position at chromosome 3p25.3. The gene is ubiquitously expressed in early development and later becomes more markedly expressed in the developing heart, limb buds, mandible and central nervous system. Expression persists in adulthood in most tissues. Sequence analysis suggests that this is a cell adhesion protein. The mouse orthologue was cloned and mapped to the syntenic region of mouse chromosome 6. Orthologues or homologues have also been identified for cow, Chinese hamster, Drosophila and Caenorhabditis elegans. The CRELD1 gene is deleted in the human cytogenetic disorder 3p- syndrome and is in the region of loss of heterozygosity for several types of cancer. A potential role for this protein in these disorders is discussed.     

Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites

The heterochronic gene lin-28 is a regulator of developmental timing in the nematode Caenorhabditis elegans. It must be expressed in the first larval stage and downregulated by the second stage for normal development. This downregulation is mediated in part by lin-4, a 21-nt microRNA. If downregulation fails due to a mutation in a short sequence in the lin-28 3' UTR that is complementary to lin-4, then a variety of somatic cell lineages fail to progress normally in development. Here, we report that Lin-28 homologues exist in diverse animals, including Drosophila, Xenopus, mouse, and human. These homologues are characterized by the LIN-28 protein's unusual pairing of RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral-type CCHC zinc knuckles. Conservation of LIN-28 proteins shows them to be distinct from the other conserved family of CSD-containing proteins of animals, the Y-box proteins. Importantly, the LIN-28 proteins of Drosophila, Xenopus, and mouse each appear to be expressed and downregulated during development, consistent with a conserved role for this regulator of developmental timing. In addition, the extremely long 3' UTRs of mouse and human Lin-28 genes show extensive regions of sequence identity that contain sites complementary to the mammalian homologues of C. elegans lin-4 and let-7 microRNAs, suggesting that microRNA regulation is a conserved feature of the Lin-28 gene in diverse animals.     

Genomic structure,conservation and FISH mapping of the Rattus norvegicus Adprt gene

Poly(ADP-ribose) polymerase 1 (PARP-1) lies at the basis of a DNA-interacting protein family that maintains genome integrity. Here we describe the genomic organisation of rat PARP-1 gene (Adprt), refine its assignment to rat chromosome (RNO) 13q25-->q26 by FISH and compare its genomic organisation between rat, mouse and human. It appears that in human, mouse and rat Adprt consists of 23 similar-sized exons with well-conserved intron and exon borders. Adprt orthologs map to homologous chromosome regions at the termini of the q-arms of human and mouse chromosomes 1 and rat 13, with gene order being conserved between the rodents. Kimura protein distance comparison with human PARP-1 as reference revealed the bovine protein to be the least conserved with 10.3 substitutions per 100 amino acids, followed by rat (8.6) and mouse (8.4).     

The myosin light chain kinase gene is not duplicated in mouse: partial structure and chromosomal localization of Mylk

The gene encoding myosin light chain kinase (MYLK) is duplicated on human chromosome 3 (HSA3; 3p13;3q21) and on a chromosome with conserved synteny to HSA3 in most non-human primate species. In human, the functional copy resides on 3q21, whereas the 3p13 site contains a pseudogene. To trace the origin of the duplication, we characterized the mouse gene Mylk. A single sequence corresponding to the functional Mylk was detected. We sequenced a 180-kb bacterial artificial chromosome clone containing the 24 first exons of Mylk; the complete mouse gene is expected to span >200 kb. Comparisons with the draft of the human genome revealed that the sequence and structure of MYLK are conserved in mammals. Fluorescence in situ hybridization (FISH) analysis indicated that the mouse gene localizes to a single site on chromosome 16B4-B5, a region with conserved synteny with HSA3q. Our study provides information on both the structure and the evolution of MYLK in mammals and suggests that it was duplicated after the divergence of rodents and primates.     

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24-->q25, 18p12-->p11, 8q32.1 and 2q22.1-->q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29-->p27, 2q112-->q113 and 2q112-->q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.     

Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis.

The human SART1 gene was initially identified in a screen for proteins recognised by IgE, which may be implicated in atopic disease. We have examined the genomic structure and cDNA sequence of the SART1 gene in the compact genomes of the pufferfish Fugu rubripes and Tetraodon nigroviridis. The entire coding regions of both the Fugu and Tetraodon SART1 genes are contained within single exons. The Fugu gene contains only one intron located in the 5' untranslated region. Southern blot hybridisation of Fugu genomic DNA confirmed the SART1 gene to be single copy. Partial genomic structures were also determined for the human, mouse, Drosophila and C. elegans SART1 homologues. The human and mouse genes both contain many introns in the coding region, the human gene possessing at least 20 exons. The Drosophila and C. elegans homologues contain 6 and 12 exons, respectively. This is only the second time such a difference in the organization of homologous Fugu and human genes has been reported. The Fugu and Tetraodon SART1 genes encode putative proteins of 772 and 774 aa, respectively, each having 65% amino acid identity to human SART1. Leucine zipper and basic motifs are conserved in the predicted Fugu and Tetraodon proteins.     

Localization of the photoreceptor gene ROM1 to human chromosome 11 and mouse chromosome 19: sublocalization to human 11q13 between PGA and PYGM.

Rom-1 is a retinal integral membrane protein that, together with the product of the human retinal degeneration slow gene (RDS), defines a photoreceptor-specific protein family. The gene for rom-1 (HGM symbol: ROM1) has been assigned to human chromosome 11 and mouse chromosome 19 by Southern blot analysis of somatic cell hybrid DNAs. ROM1 was regionally sublocalized to human 11p13-11q13 by using three mouse-human somatic cell hybrids; in situ hybridization refined the sublocalization to human 11q13. Analysis of somatic cell hybrids suggested that the most likely localization of ROM1 is in the approximately 2-cM interval between human PGA (human pepsinogen A) and PYGM (muscle glycogen phosphorylase). ROM1 appears to be a new member of a conserved syntenic group whose members include such genes as CD5, CD20, and OSBP (oxysterol-binding protein), on human chromosome 11 and mouse chromosome 19. Localization of the ROM1 gene will permit the examination of its linkage to hereditary retinopathies in man and mouse.     

Deoxyribonuclease II: structure and chromosomal localization of the murine gene, and comparison with the genomic structure of the human and three C. elegans homologs

Deoxyribonuclease II (DNase II) has been implicated in diverse functions including degradation of foreign DNA, genomic instability, and in mediating the DNA digestion associated with apoptosis. The production of a mouse deleted for DNase II would clearly help to discriminate these functions. We have cloned and sequenced the mouse gene encoding DNase II. It was found to have a similar intron/exon structure to the human gene, although introns 3 and 5 are considerably shorter. The gene is located on mouse chromosome 8. The order of genes at this locus is mGCDH, mEKLF, mDNase II, mSAST, which is the same order that these genes are found on human chromosome 19. The GenBank database contains incorrect expressed sequence tags (ESTs) for the 3' end of the mouse mRNA. Furthermore, the gene structure of two of the three homologs in C. elegans is also incorrectly predicted in the database. We have established the correct intron/exon structure for these genes and show the conserved sequence and structure of the C. elegans, murine and human genes.     

Conserved autosomal syntenic group on mouse (MMU) chromosome 15 and human (HSA) chromosome 22: assignment of a gene for arylsulfatase A to MMU 15 and regional mapping of DIA1, ARSA, and ACO2 on HSA 22

We have utilized a panel of Chinese hamster x mouse somatic cell hybrids segregating mouse chromosomes to assign a gene for arylsulfatase A (ARSA) to mouse chromosome 15. Considering our previous assignment of a gene for diaphorase-1 (DIA1) to the same mouse chromosome, we have evidence for another syntenic relationship that has been conserved, since the homologous loci for human ARSA and DIA1 are both located on human chromosome 22. Because MMU 15 and HSA 22 are quite dissimilar in size and banding patterns, we have attempted to identify the conserved portion by regional mapping of human DIA1 and ARSA using somatic cell hybrids segregating a human chromosome translocation t(15;22)(q14;q13.31). The results assign human DIA1 and ARSA to the distal sub-band of 22q13 (region 22q13.31 leads to qter). The locus for mitochondrial aconitase (ACO2) has been separated by the breakpoint from DIA1 and ARSA and is located more proximally.     

A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues

Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse x hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR(5000). The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.     

Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes.

Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.     

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

UBL5 is essential for pre‐mRNA splicing and sister chromatid cohesion in human cells

UBL5 is an atypical ubiquitin‐like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre‐mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre‐mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre‐mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.