The Human GCAP1 and GCAP2 Genes Are Arranged in a Tail-to-Tail Array on the Short Arm of Chromosome 6 (p21.1)

GCAP1 and GCAP2 are related Ca²⁺-binding proteins that activate photoreceptor guanylate cyclase(s). We showed previously that the human GCAP1 gene, consisting of four exons, is located at 6p21.1 (locus designation GUCA). To identify the chromosomal location of the GCAP2 gene, we first cloned its cDNA and determined its intron–exon distribution by PCR analysis. The results show that the introns of the GCAP2 gene are positioned exactly as in the GCAP1 gene and are nearly double in size. Sequence similarity between the two genes, however, is limited to portions of exons 1 and 2. The GCAP1 and GCAP2 genes are transcribed into single mRNA species (1.7 and 2.2 kb, respectively) and are detectable only in the retina by Northern blotting. The GCAP2 gene was found by somatic human–hamster hybrid panel analysis and FISH to reside at GUCA in a region indistinguishable from that of GCAP1. PCR analysis with exon 4-specific primers showed that the genes are in a tail-to-tail array less than 5 kb apart and altogether span less than 20 kb of genomic DNA. The identical gene structures and loci of GCAP1 and GCAP2, and the identical function of the gene products, are consistent with a gene duplication event.     

Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes)

The guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins of the calmodulin (CaM) gene superfamily that function in the regulation of photoreceptor guanylate cyclases (GCs). In the mammalian retina, two GCAPs (GCAP 1-2) and two transmembrane GCs have been identified as part of a complex regulatory system responsive to fluctuating levels of free Ca(2+). A third GCAP, GCAP3, is expressed in human and zebrafish (Danio rerio) retinas, and a guanylate cyclase-inhibitory protein (GCIP) has been shown to be present in frog cones. To explore the diversity of GCAPs in more detail, we searched the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) genomes for GCAP-related gene sequences (fuGCAPs and zGCAPs, respectively) and found that at least five additional GCAPs (GCAP4-8) are predicted to be present in these species. We identified genomic contigs encoding fuGCAPl-8, fuGCIP, zGCAPl-5, zGCAP7 and zGCIP. We describe cloning, expression and localization of three novel GCAPs present in the zebrafish retina (zGCAP4, zGCAP5, and zGCAP7). The results show that recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca(2+)-dependent manner. RT-PCR with zGCAP specific primers showed specific expression of zGCAPs and zGCIP in the retina, while zGCAPl mRNA is also present in the brain. In situ hybridization with anti-sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. The presence of at least eight GCAP genes suggests an unexpected diversity within this subfamily of Ca(2+)-binding proteins in the teleost retina, and suggests additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4-8 or GCIP genes.     

Identification of a Ferritin Light Chain Pseudogene Near the Glycerol Kinase Locus in Xp21 by cDNA Amplification for Identification of Genomic Expressed Sequences

We used cDNA amplification for identification of genomic expressed sequences (CAIGES) to identify genes in the glycerol kinase region of the human X chromosome. During these investigations we identified the sequence for a ferritin light chain (FTL) pseudogene in this portion of Xp21. A human liver cDNA library was amplified by vector primers, labeled, and hybridized to Southern blots ofEcoRIdigested human genomic DNA from cosmids isolated from yeast artificial chromosomes in the glycerol kinase region of Xp21. A 3.1-kb restriction fragment hybridized with the cDNA library, was subcloned and sequenced, and a 440-bp intronless sequence was found with strong similarity to the FTL coding sequence. Therefore, the FTL pseudogene that had been mapped previously to Xp22.3–21.2 was localized specifically to the glycerol kinase region. The CAIGES method permits rapid screening of genomic material and will identify genomic sequences with similarities to genes expressed in the cDNA library used to probe the cloned genomic DNA, including pseudogenes.     

Structure and Ca2+ regulation of frog photoreceptor guanylate cyclase,ROS-GC1

Rod outer segment membrane guanylate cyclase (ROS-GC) is a critical component of the vertebrate phototransduction machinery. In response to photoillumination, it senses a decline in free Ca²⁺ levels from 500 to below 100 nM, becomes activated, and replenishes the depleted cyclic GMP pool to restore the dark state of the photoreceptor cell. It exists in two forms, ROS-GC1 and ROS-GC2. In outer segments, ROS-GCs sense fluctuations in Ca²⁺ via two Ca²⁺-binding proteins, which have been termed GCAP1 and GCAP2. In the present study we report on the cloning of two ROS-GCs from the frog retinal cDNA library. These cyclases are the structural and functional counterparts of the mammalian ROS-GC1 and ROS-GC2. There is, however, an important difference between the regulation of mammalian and frog ROS-GC1: In contrast to the mammalian, the frog form does not require the myristoylated form of GCAP1 for its Ca²⁺-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca²⁺ because unmyristoylated GCAP1 mutants which do not bind Ca²⁺, activate frog ROS-GC1. The findings establish frog as a suitable phototransduction model and show a facet of frog ROS-GC signaling, which is not shared by the mammalian form.     

Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21.3) by cDNA hybridization selection.

It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC.     

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6–7 kb from C9 in a head-to-head or 5′ to 5′ orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.     

Localization of a new prostate-specific antigen-related serine protease gene, KLK4, is evidence for an expanded human kallikrein gene family cluster on chromosome 19q13.3-13.4.

The human tissue kallikrein (KLK) family of serine proteases, which is important in post-translational processing events, currently consists of just three genes-tissue kallikrein (KLK1), KLK2, and prostate-specific antigen (PSA) (KLK3)-clustered at chromosome 19q13. 3-13.4. We identified an expressed sequence tag from an endometrial carcinoma cDNA library with 50% identity to the three known KLK genes. Primers designed to putative exon 2 and exon 3 regions from this novel kallikrein-related sequence were used to polymerase chain reaction-screen five cosmids spanning 130 kb around the KLK locus on chromosome 19. This new gene, which we have named KLK4, is 25 kb downstream of the KLK2 gene and follows a region that includes two other putative KLK-like gene fragments. KLK4 spans 5.2 kb, has an identical genomic structure-five exons and four introns-to the other KLK genes and is transcribed on the reverse strand, in the same direction as KLK1 but opposite to that of KLK2 and KLK3. It encodes a 254-amino acid prepro-serine protease that is most similar (78% identical) to pig enamel matrix serine protease but is also 37% identical to PSA. These data suggest that the human kallikrein gene family locus on chromosome 19 is larger than previously thought and also indicate a greater sequence divergence within this family compared with the highly conserved rodent kallikrein genes.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

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.     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

The crystal structure of GCAP3 suggests molecular mechanism of GCAP-linked cone dystrophies

Absorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic-GMP (cGMP). This hydrolysis promotes the closing of cGMP-gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate cyclase-activating proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca2+-dependent manner. At high [Ca2+], typical of the dark-adapted state (approximately 500 nM), GCAPs inhibit retinal GCs. At the low [Ca2+] (approximately 50 nM) that occurs after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark-state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca2+ bound. GCAP3 is an EF-hand Ca2+-binding protein with Ca2+ bound to EF2, 3 and 4, while Ca2+ binding to EF-hand 1 is disabled. GCAP3 contains two domains with the EF-hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca2+-binding proteins. Residues not involved in Ca2+ binding, but conserved in all GCAPs, cluster around EF1 in the N-terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations.     

Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

Organization of the human genes for insulin-like growth factors I and II

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.