Taken collectively, our transfection and immuno-analyses offered new information regarding 16 genes in the protein and cellular amounts (Figs

Taken collectively, our transfection and immuno-analyses offered new information regarding 16 genes in the protein and cellular amounts (Figs. on mouse genes, we looked the human BGB-102 being genome data source for orthologs. Human being orthologs for 13 mouse genes had been within genomic parts of conserved synteny between human beings and mice. The additional 11 genes didn’t have human being orthologs, recommending differential expansion in the mouse genome. The protein-coding region of each gene was defined by selecting the longest amino acid sequence terminating before a polyadenylation signal (if there is one present), and deduced amino acid sequences were subjected to database searches. Nineteen gene products were predicted to contain various domains and motifs, and found to be annotated with gene ontology codes. Thus, based on the em in silico /em information, some of these proteins are predicted to be implicated in transcriptional regulation and/or nuclear activity (Mm.290718, Mm.157767, Mm.85045, Mm.86671 Mm.373242, Mm.437189 and “type”:”entrez-nucleotide”,”attrs”:”text”:”Mm116803″,”term_id”:”1531388630″,”term_text”:”MM116803″Mm116803), metabolic processes (Mm.46159, Mm.55870, Mm.333010, Mm.252733, Mm86671 and Mm.159795) and cell structure (Mm.45833 and Mm.23534) (Table ?(Table44). Open in a separate window Figure 4 Genomic, transcript, and protein characteristics of the genes in silico. Gene structure and exon organization were determined using genome database searches. In exon organization, the boxes represent exons. The bars indicate regions amplified in the PCR analysis and used as probes in the Northern blot analysis. Coding regions were determined by selecting the longest open reading frames deduced from cDNA sequences. The predicted coding regions are shaded. The position of the poly A signal is marked by filled arrowheads, and the presence of poly A is indicated by ‘A’. Chromosomal location was determined by searching the mouse and human genome databases. The predicted amino acid sequences of genes were analyzed using various bioinformatics tools (see Experimental Procedure). For annotation of genes with ontology terms, amino acid sequences were submitted to and subsequently obtained from exclusive web servers (Goblet), which use a variety of different protein databases and provide BGB-102 gene ontology codes. Each gene ontology code falls into one of the larger categories of molecular function (M), cellular component (C), or biological process (B). Table 4 Putative functions of the eight gene products in reproduction thead FunctionUniGene em In silico /em informationLocalization in GC-2 cellsPresence in TSCPresence in MS /thead Transcriptional regulationMm.290718TranscriptionNun.d.n.d.Mm.86671DNA bindingNun.d.n.d.Mm.373242TranscriptionNun.d.n.d.Nuclear integrityMm.437189Perinuclear proteinNun.d.n.d.Sperm structure or motilityMm.23534Tektin3Cy++ (T)Mm.23377Tep22n.d.++Mm.159795CatSper3ERn.d.n.d.FertilizationMm.333010Trypsin-like serine proteaseGA++ (A) Open in a separate window Eight genes with em in vitro /em protein data (Figs. 5, 6 and 7) which support em in silico /em information (Table 1 and Figure 4) are functionally categorized. Abbreviations are as follows: TSC, testicular spermatogenic cells; MS, mature sperm; Nu, nucleus; Cy, cytoplasm; ER, endoplasmic reticulum GA, Golgi apparatus; n.d., not determined; (T), tail; (A), acrosome. BGB-102 Subcellular localization of the proteins To explore protein characteristics em FEN1 in vitro /em , we investigated subcellular localization of the gene products [14]. GFP-tagged full-length gene sequences were transiently transfected into GC-2 cells. GC-2 cells are immortalized germ cells (spermatocytes) of mouse testis [15]. We observed GFP signals from 14 out of the 24 gene products analyzed. By contrast, the GFP signals were not detected in the other 10 genes, suggesting that the expression of these proteins is highly transient, very low in amount or delayed. Figure ?Figure55 depicts the subcellular locations identified. Five gene products were found to be localized in the nucleus. Other gene products localized to the endoplasmic reticulum (three genes), Golgi apparatus (three genes), and cytoplasm (three genes). It should be noted that the three genes with cytoplasmic localization displayed a speckled localization.