Genomic organization of the human galpha14 and Galphaq genes and mutation analysis in chorea-acanthocytosis (CHAC)

Chorea-acanthocytosis (CHAC) (OMIM 200150) is a rare neurological syndrome characterized by neurodegeneration in combination with morphologically abnormal red cells (acanthocytosis). A partial yeast artificial chromosome contig of the CHAC critical region on chromosome 9q21 has been constructed, and 21 expressed sequence tags have been mapped. We have subsequently cloned Galpha14, a member of the G-protein alpha-subunit multigene family, and have identified Galphaq in the contig. The genomic structure of both genes has been established after construction of a bacterial artificial chromosome contig that showed Galphaq and Galpha14 to be in a head-to-tail arrangement (Cen-Galphaq-Galpha14-qter). Northern analysis found Galphaq to be ubiquitously expressed and Galpha14 to display a more restricted pattern of expression. Mutation analysis of the coding regions and splice sites for Galphaq and Galpha14 in 10 affected individuals from different families identified no changes likely to cause disease; however, two distinct single nucleotide polymorphisms in the coding region of Galpha14 have been identified. This study has excluded two plausible candidate genes from involvement in CHAC and has provided a solid platform for a positional cloning initiative.     

Genomic organization of the human retinoic acid receptor beta 2.

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.     

Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.     

Genomic organization of human lactate dehydrogenase-A gene.

A human genomic clone containing the lactate dehydrogenase-A (LDH-A) gene of approx. 12 kilobases in length was isolated and characterized. The protein-coding sequence is interrupted by six introns, and the positions of these introns are at the random coil regions or near the ends of secondary structures located on the surface of the LDH-A molecule. An additional intron is present at 24 nucleotides 5' to the translation initiation codon ATG, while the 3' untranslated sequence of 565 nucleotides is not interrupted. The genomic blot analysis of human placenta DNA indicates the presence of multiple LDH-A gene-related sequences.     

Genomic organization and recombination analysis of human norovirus identified from China

Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis in both industrial and developing countries including China. Recent studies have revealed that NoV genome is highly prone to mutation and recombination which may lead to emergence of new strains. In the present study, three full-length genomes of human NoV from China were determined and the genomic organization and recombination were analyzed. They had similar genome organization and contained three predicted ORFs, though the 5′UTR of those three strains were 2, 4 and 8 nucleotides, respectively. Phylogenetic analysis showed that the HU/GII/SHANGHAI/SH312/2008/CHN strain may be a recombinant of GII-3 capsid and GII-4 polymerase. To confirm the finding and detect the breakpoints where the recombination event occurred, we performed recombination analysis based on the genomic sequences of HU/GII/SHANGHAI/SH312/2008/CHN as the query sequence, and AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3 as the background sequences, using RPD software. Results indicated that the two parental strains were AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3. The breakpoint for this recombination event located at position 5,107 nt of the genome (in the ORF1 and ORF2 overlap).     

Genomic organization and functional analysis of murine PKD2L1

Mutations in genes that encode polycystins 1 or 2 cause polycystic kidney disease (PKD). Here, we report the genomic organization and functional expression of murine orthologue of human polycystin-2L1 (PKD2L1). The murine PKD2L1 gene comprises 15 exons in chromosome 19C3. Coexpression of PKD2L1 together with polycystin-1 (PKD1) resulted in the expression of PKD2L1 channels on the cell surface, whereas PKD2L1 expressed alone was retained within the endoplasmic reticulum (ER). This suggested that interaction between PKD1 and PKD2L1 is essential for PKD2L1 trafficking and channel formation. Deletion analysis at the cytoplasmic tail of PKD2L1 revealed that the coiled-coil domain was important for trafficking by PKD1. Mutagenesis within two newly identified ER retention signal-like amino acid sequences caused PKD2L1 to be expressed at the cell surface. This indicated that the coiled-coil domain was responsible for retaining PKD2L1 within the ER. Functional analysis of murine PKD2L1 expressed in HEK 293 cells was undertaken using calcium imaging. Coexpression of PKD1 and PKD2L1 resulted in the formation of functional cation channels that were opened by hypo-osmotic stimulation, whereas neither molecule formed functional channels when expressed alone. We conclude that PKD2L1 forms functional cation channels on the plasma membrane by interacting with PKD1. These findings raise the possibility that PKD2L1 represents the third genetic locus that is responsible for PKD.     

Genomic organization and localization of the human CRMP-1 gene.

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein considered to be involved in the collapsin-induced growth cone collapse during neural development. CRMP-1 belongs to the Unc-33 gene family. Here we report the genomic structure and the localization of the human CRMP-1 gene to chromosome 4p16.1. Sequence analysis revealed that the human CRMP-1 gene consists of 14 exons. We have also established sequencing assays for all its coding exons. This should permit the rapid screening for mutations to assess CRMP-1 role in genetic disorders mapped in the 4p16.1 region.     

Genomic organization and comparative sequence analysis of the mouse and human FRS2, FRS3 genes

The signaling adapter proteins FRS2 and FRS3 are implicated in the transmission of extracellular signals from nerve growth factor (NGF) or fibroblast growth factor (FGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. This study presents the genomic sequence and exon-intron organization of the mouse FRS2 and FRS3 loci as well as their evolutionary conservation with their human counterparts. Both FRS2 and FRS3 contain 5 coding exons spanning over 7 kb of genomic sequence with similar exon sizes and organization. Comparative genomic sequence analyses show a highly conserved genomic organization between mouse and human in both FRS2 and FRS3 genes. Non-coding sequences, highly conserved between mouse and human, were identified in the FRS3 introns that may potentially function as regulatory elements. To assay potential differences in their patterns of expression, RT-PCR analysis was used to assay FRS2 and FRS3 expression in the developing embryo and neural tube (NT) during the time of neurogenesis.     

Genomic organization of the sheep immunoglobulin JH segments and their contribution to heavy chain variable region diversity

 The sheep immunoglobulin heavy chain Igh-J locus has been characterized in order to determine the genomic organization of JH segments and their contribution to heavy chain diversity. The locus contains six segments, of which two are functional and four are apparently pseudogenes. These segments span a 1.8 kilobase (kb) region. The distance between JH-ps4 (the 3′-most segment) and the first domain of the μ-chain encoding constant gene is about 5 kb. The two functional JH segments have a standard upstream recombination signal sequence, including heptamer and nonamer sequences separated by a 22–23 nucleotide spacer, and end with a RNA donor splice site. These two segments possess all the characteristic JH invariant residues and are found in expressed μ heavy chain variable regions. The 5′ functional JH1 segment is used in more than 90% of the cDNAs sequenced to date. The contribution of JH segment germline multiplicity to variable regions diversity appears therefore to be minimal. Comparison with other mammalian JH segments shows that all loci are very closely related and probably have evolved from a common ancestral locus. Received: 19 November 1996 / Revised: 17 March 1997     

Genomic organization and chromosomal localization of the human CD27 gene.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.