Testis fascin (FSCN3): a novel paralog of the actin-bundling protein fascin expressed specifically in the elongate spermatid head

During spermiogenesis, significant morphological changes occur as round spermatids are remodeled into the fusiform shape of mature spermatozoa. These changes are correlated with a reorganization of microfilaments and microtubules in the head and tail regions of elongating spermatids. There is also altered expression of specialized actin- and tubulin-associated proteins. We report the characterization of a novel, spermatid-specific murine paralog of the actin-bundling protein fascin (FSCN1); this paralog is designated testis fascin or FSCN3. Testis fascin is distantly related to fascins but retains its primary sequence organization. cDNA clones of mouse testis fascin predict a 498 amino acid protein of molecular mass 56 kD that shares 29% identity with mouse fascin. Mapping of murine and human FSCN3 genes shows localization to the 7q31.3 chromosome. Northern analysis indicates that FSCN3 expression is highly specific to testis and that in situ hybridization further restricts expression to elongating spermatids. Antibodies raised against recombinant FSCN3 protein identify a band at 56 kD in testis, epididymis, and epididymal spermatozoa, suggesting that testis fascin persists in mature spermatozoa. In accord with the in situ hybridization results, immunofluorescent microscopy localizes testis fascin protein to areas of the anterior spermatid head that match known distributions of F-actin in the dorsal and ventral subacrosomal spaces. It is possible that testis fascin may function in the terminal elongation of the spermatid head and in microfilament rearrangements that accompany fertilization.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

人和大鼠自噬相关新基因WIPI- 3 的电子克隆和序列分析

目的：人和大鼠WIPI-3基因的电子克隆和序列分析。方法：综合运用基因电子拼接和比较基因组分析等生物信息学手段进行新基因的电子克隆和注释。通过拼接CR593190、NM 019613和BC00097 cDNA序列获得人WIPI-3 cDNA全长序列,通过拼接EST序列CB727439、CB737031、BF557312、BG663387和预测的CDS获得大鼠WIPI3基因的cDNA序列。结果：成功克隆了人和大鼠自噬相关新基因WIPI-3的cDNA,上述两个新基因序列均得到了人类基因组序列和EST序列的双重支持,另外经RT-PCR验证电子克隆的基因序列也是正确的,而且序列均已被GenBank收录,其编号分别为：AM182326和NM-001039587。其中,人WIPI-3基因修正了目前基因库中注释的WIPI-3序列,而大鼠基因则是国际上首次克隆,井被确定为参考序列。结论：基因电子拼接结合种属同源基因比较分析,可大大提高电子克隆的精确性,这种方法可有效用于新基因的克隆和注释。     

The isolation,characterisation, and chromosomal assignment of the gene for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, (HMG-CoA reductase)

Summary We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12.     

Role of fascin in filopodial protrusion

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t(1/2) < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.     

Mechanism of actin filament bundling by fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ～5 nm apart, resulting in a distance between actin filaments in the bundle of ～8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.     

cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.     

Intrinsic dynamic behavior of fascin in filopodia

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

The Human Prohibitin (PHB) Gene Family and Its Somatic Mutations in Human Tumors

Five cosmid clones, isolated by procedures to screen genomic libraries for homologous variants of the human prohibitin gene (PHB), were analyzed to determine their genomic structures. Four of these (PHBP1-4) were found to be processed pseudogenes, each located on a different chromosome from their counterparts on chromosome 17q21. The DNA sequence of one clone (PHBP1, on chromosome 6q25) shared a 91.3% identity at the nucleotide level with the cDNA of functional prohibitin. A large number of human tumors of the breast, ovary, liver, and lung were examined for somatic mutations in the PHB gene. Although mutations were observed in a few sporadic breast cancers, none were identified in any of the other cancers.     

Cloning of the human monocarboxylate transporter MCT3 gene: localization to chromosome 22q12.3-q13.2.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

The zebrafish fth1, slc3a2, men1, pc, fgf3 and cycd1 genes define two regions of conserved synteny between linkage group 7 and human chromosome 11q13

In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.     

The retinal fascin gene 2 (FSCN2) — partial structural analysis and polymorphism detection in dogs with progressive retinal atrophy (PRA)

The retinal fascin 2 gene (FSCN2) underwent a molecular analysis, a search for polymorphisms and an evaluation as a candidate gene for retinopathies in dogs. Specific fragments of the gene encompassing partial exon 1 and intron 1, and exons 2-5 with respective introns were sequenced and these data were deposited in the GenBank database. Three distinct polymorphic sites detectable with PCR-RFLP were found--AM050719: g.237G>A, AM050719: g.525A>G, and AM050720: g.1071A>G. No positive associations between these polymorphisms and the PRA-clinical status were observed in the investigated population consisting of Poodles, American Cocker Spaniels, and English Cocker Spaniels. In spite of that, the FSCN2 gene remains an excellent candidate gene for retinopathies in dogs and the results can contribute to further research in this field.