Germline Specificity Research Articles

Construction of a germline-specific RNAi tool in C. elegans

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and ageing. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi activities in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis

Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype of Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In transgenic Arabidopsis (Arabidopsis thaliana), PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production, consistent with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding motifs are identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of previously reported Germline Restrictive Silencer Factor binding sequence. Instead, a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.

Improved Efficiency of Female Germline Stem Cell Purification Using Fragilis-Based Magnetic Bead Sorting

The enrichment of female germline stem cells (FGSCs) and the establishment of cell lines are influenced by the efficiency of cell purification. A previous study using mouse vasa homolog (MVH)-magnetic bead sorting for the isolation and purification of mouse FGSCs showed a relatively low efficiency. In this study, we tested 3 further proteins with the aim of improving the efficiency of FGSC purification. Immunofluorescence assays and magnetic sorting were performed using short-type pituitary gland and brain-cadherin (Stpb-c), CD9, and interferon-inducible transmembrane protein 3 (Iftm3, Fragilis), all of which are expressed in germ cells. Although all 3 proteins were expressed in FGSCs, CD9 was unsuitable because of its lack of germline specificity, and Stpb-c was also unsuitable because of the unavailability of an appropriate primary antibody. The efficiency of FGSC purification was remarkably enhanced using the germline-specific protein Fragilis, compared with that using MVH. This new method for the purification of FGSCs may have extensive applications in stem cell studies and clinical research.

Sequence and expression pattern of the germ line marker vasa in honey bees and stingless bees

Queens and workers of social insects differ in the rates of egg laying. Using genomic information we determined the sequence of vasa, a highly conserved gene specific to the germ line of metazoans, for the honey bee and four stingless bees. The vasa sequence of social bees differed from that of other insects in two motifs. By RT-PCR we confirmed the germ line specificity of Amvasa expression in honey bees. In situ hybridization on ovarioles showed that Amvasa is expressed throughout the germarium, except for the transition zone beneath the terminal filament. A diffuse vasa signal was also seen in terminal filaments suggesting the presence of germ line cells. Oocytes showed elevated levels of Amvasa transcripts in the lower germarium and after follicles became segregated. In previtellogenic follicles, Amvasa transcription was detected in the trophocytes, which appear to supply its mRNA to the growing oocyte. A similar picture was obtained for ovarioles of the stingless bee Melipona quadrifasciata, except that Amvasa expression was higher in the oocytes of previtellogenic follicles. The social bees differ in this respect from Drosophila, the model system for insect oogenesis, suggesting that changes in the sequence and expression pattern of vasa may have occurred during social evolution.

Concentration dependent selection of targets by an SR splicing regulator results in tissue-specific RNA processing

The splicing factor Transformer-2 (Tra2) is expressed almost ubiquitously in Drosophila adults, but participates in the tissue-specific regulation of splicing in several RNAs. In somatic tissues Tra2 participates in the activation of sex-specific splice sites in doublesex and fruitless pre-mRNAs. In the male germline it affects splicing of other transcripts and represses removal of the M1 intron from its own pre-mRNA. Here we test the hypothesis that the germline specificity of M1 repression is determined by tissue-specific differences in Tra2 concentration. We find that Tra2 is expressed at higher levels in primary spermatocytes of males than in other cell types. Increased Tra2 expression in other tissues reduces viability in a manner consistent with known dose-dependent effects of excessive Tra2 expression in the male germline. Somatic cells were found to be competent to repress M1 splicing if the level of Tra2 transcription was raised above endogenous concentrations. This suggests not only that M1 repression is restricted to the germline by a difference in Tra2 transcription levels but also that the protein's threshold concentration for M1 regulation differs from that of doublesex and fruitless RNAs. We propose that quantitative differences in regulator expression can give rise to cell-type-specific restrictions in splicing.

Identification of a cis-Acting Sequence Required for Germ LineSpecific Splicing of the P Element ORF2-ORF3 Intron

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.

Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron.

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.

Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron.

P-element transposition is limited to the germ line because the element's third intron is only spliced in germ line cells. We show that a 240-bp fragment containing this 190-bp intron can confer germ line specificity when placed in the context of another gene. We find that the cis-acting regulatory sequences required for germ line regulation map near to, but not at, the 5' or 3' splice junctions.

Somatic effects of P element activity in Drosophila melanogaster: pupal lethality.

Nonautonomous P elements normally excise and transpose only when a source of transposase is supplied, and only in the germline. The germline specificity depends on one of the introns of the transposase gene which is not spliced in somatic cells. To study the effects of somatic P activity, a modified P element (delta 2-3) lacking this intron was used as a source of transposase. Nonautonomous P elements from a strain called Birmingham, when mobilized in somatic cells by delta 2-3, were found to cause lethality, although neither component was lethal by itself. The three major Birmingham chromosomes acted approximately independently in producing the lethal effect. This lethality showed a strong dependence on temperature. Although temperature sensitivity was limited to larval stages, the actual deaths occurred at the pupal stage. Survivors, which could be recovered by decreasing the temperature or by reducing the proportion of the Birmingham genome present, often showed multiple developmental anomalies and reduced longevity reminiscent of the effects of cell death from radiation damage. Although the genetic damage occurred in dividing imaginal disc cells, the phenotypic manifestations--death and abnormalities--are not observed until later. The survivors also showed gonadal dysgenic (GD) sterility, a well-known characteristic of P-M hybrid dysgenesis. To explain these findings, we suggest that pupal lethality and GD sterility are both caused by massive chromosome breakage in larval cells, resulting from excision and transposition of genomic P elements acting as substrate for the transposase.

Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans.

We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.

Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing

We show that the germline specificity of P element transposition is controlled at the level of mRNA splicing and not at the level of transcription. In the major P element RNA transcript, isolated from somatic cells, the first three open reading frames are joined by the removal of two introns. Using in vitro mutagenesis and genetic analysis we demonstrate the existence of a third intron whose removal is required for transposase production. We propose that this intron is only removed in the germline and that its removal is the sole basis for the germline restriction of P element transposition.

Germ line specificity of P-element transposition and some novel patterns of expression of transduced copies of the white gene.

Germ line specificity of P-element transposition and some novel patterns of expression of transduced copies of the white gene.

From the mechanisms of lymphocyte activation to internal activity in the immune system

The principles of lymphocyte activation were summarized for all three sets of immunocompetent cells: B cells, cytotoxic cells and helper T lymphocytes. They were then used to derive the basic mechanisms and specificities which drive internal activity in the normal immune system and which select available antibody repertoires. It was postulated that "natural antibodies" are induced by "natural helper cells" and are selected on the basis of their idiotypic profiles, which are complementary to available T-helper-cell repertoires. "Natural idiotypes" would then carry idiotopes similar to MHC epitopes, the predominant germ-line specificity of T-cell repertoires.

Late clonal selection and expansion of the TEPC-15 germ-line specificity.

The maturation of the antibody response to phosphorylcholine (PC) in neonatal BALB/c mice was studied. A T cell-independent class 1 1 PC-antigen, 3-(p-azophenyl phosphorylcholine)-N-acetyl-L-tyrosylglycylglycine lipopolysaccharide, was synthesized and used to immunize neonatal mice of different ages. The earliest anti-PC hemolytic plaque-forming response could be induced in 1-d-old neonates. Idiotype analysis on these early anti-PC antibodies showed that the response was not TEPC-15 dominant although TEPC-15-positive plaque-forming cells were detected. However, idiotype analysis of the anti-PC-LPS response in 7 d or older animals indicated that clonal dominance had been established. Similar results were obtained in splenic fragment culture with cells from neonatal livers and spleens. PC-specific precursors were detected in the liver of 1-d-old neonates, whereas the spleen of those animals contained no precursors for PC. Precursors for PC residing in the neonatal liver are not TEPC-15 dominant, whereas the splenic PC precursors of 5- to 6-day-old animals express the TEPC-15 idiotype dominatly. These findings demonstrate that during ontogeny PC-specific B cells appear before the TEPC-15 clone becomes dominant. Thus clonal dominance in the adult anti-PC response and late acquisition of the TEPC-15 specificity during ontogeny do not signify a particularly unique or direct relationship to the expression of genes encoded in the germline.