Germline

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to

The classic view of biological inheritance of traits from parent to offspring implicates genetic factors, but it is now recognized that non-genetic factors -that is, factors that modify gene activity without changing the DNA sequence and that are sensitive to the environment -also contribute. Evidence for germline-dependent non-genetic inheritance of acquired traits in mammals has accumulated in neuroscience, behavioural neuroendocrinology, environmental toxicology and nutritional science 1,2 . This mode of inheritance is crucial for medical genetics because it helps to explain the aetiology, expression and heritability of prevalent neuropsychiatric diseases such as depression, personality disorders, anxiety and autism 3 . Identification of non-genetic marks and mechanisms might provide biomarkers for disease diagnosis and treatment monitoring, and possibly prevention 4 .

Hereditary diffuse gastric cancer (HDGC) is a recently defined cancer syndrome caused by inactivating, heterozygous germline mutations in the gene for the cell-to-cell adhesion protein E-cadherin (CDH1). Here, we describe the search for CDH1 mutations in 10 newly identified gastric cancer families. Seven of 10 families met the clinical criteria for HDGC. Germline mutations were identified in four of these seven families and one family that was borderline for the clinical criteria. Of the mutations identified in the five new families, four were previously unreported and consisted of two frameshift and two donor splice site mutations. One splice site mutation occurred at the 100% conserved +1 position. The second splice site mutation occurred at the +5 position and was shown to lead to abnormal splicing. Additional CDH1 variants detected include the heterozygous 160 C®A promoter polymorphism, which has previously been reported to be associated with decreased CDH1 transcription. We, however, found this polymorphism to be common in a control population, suggesting that a major role for this polymorphism in gastric cancer susceptibility is unlikely. Hum Mutat 19:518525, 2002.

I made a sculpture of Uranus' castrated penis. Apparently, Cronus cut off his father's penis and threw it in the ocean, in the Peloponnesian Sea. He cut off his father's penis because his mother gave birth to a-hundred-handed titan, like a centipede human, and Uranus was so grossed out by the mutant with a hundred hands, that he kept that being, that person, his child, inside a cave inside of a castle. Even some people say he pushed it back up into his wife Gaia's vagina, at which point she told her son Cronus, " Why don't you go cut his dick off? " And he did. He sliced off his father's penis and he threw it in the sea, and from the foam and blood of the leaking, castrated penis of Uranus came Aphrodite, the goddess of beauty. It's a strange myth. I didn't know really much about it. I had gone for the centipede thing because I thought, " Well, maybe I'll make a person with a hundred hands someday. " But every time someone chooses someone else to make a baby with, they're making a freak for their own pleasure. It doesn't matter if it's random recombination, it doesn't matter if it's based on love, it doesn't matter if this being is cared for, sent to a really nice preschool , or fed well; or fed a bunch of crap. What matters is reproduction itself is kind of like a freak show. You want people to see your display of fertility. You want people to see that you have the wealth to go ahead and procreate. You want people to see your child perform a piano recital. Anyone would be able to show off that your genes were worthy of getting to the next generation. It's really kind of sad. Breeding is narcissism. It's like your little mirror, right? I was in a meeting that was talking about the ethics of genetic modification, and they asked parents to raise their hand if they would be willing to pay an extra five or ten thousand dollars to have children with guaranteed perfect pitch, so that they would, you know, basically not need to pay for all those piano lessons. And most parents were like, " Yeah. " That's cheaper in the long run, and that's partly what they were thinking; but they were also thinking, " I'd like a kid who's a good musician. " But I do know when people go for genetic counseling, if they were offered not only to eliminate negative traits but to add positive traits, as long as you put a price tag on it, someone's gonna hit Buy It Now. It's the other side of the mountain effect, and it's what will drive personal genetic alteration.

Mutations in the gene TP53, which codifies the tumor suppressor protein p53, are found in about 50% of tumors. These mutations can occur not only at somatic level, but also in germline. Pediatric cancer patients, mostly with additional family history of malignancy, should be considered as potential TP53 germline mutation carriers. Germline TP53 mutations and polymorphisms have been widely studied to determine their relation with different tumors' pathogenesis. Our aim was to analyze the occurrence frequency of germline TP53 mutations and polymorphisms and to relate these to tumor development in a pediatric series. Peripheral blood mononuclear cell samples from 26 children with solid tumors [PST] and 21 pediatric healthy donors [HD] were analyzed for germline mutations and polymorphisms in TP53 gene spanning from exon 5 to 8 including introns 5 and 7. These PCR amplified fragments were sequenced to determine variations. A heterozygous mutation at codon 245 was found in 1/26 PST and 0/21 HD. Comparative polymorphisms distribution, at position 14181 and 14201(intron 7), between HD and PST revealed a trend of association (p= 0.

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We

Germline development and function engages genes that are conserved in all sexually reproducing metazoans including the Piwi family genes 1-3 and genes encoding Tudor domain-containing proteins (Tdrds) 4,5 that are arranged in functional networks at a genetic and biochemical level . Piwi proteins and Tdrds colocalize in germ granules, which are cytoplasmic, electron-dense structures such as the intermitochondrial cement (IMC) of spermatocytes and chromatoid bodies of spermatids in mammals 5,10 . Piwi proteins bind piRNAs to form pi-ribonucleoproteins (piRNPs) . piRNAs have been implicated in transposon control, and a mechanism for amplification of secondary, retrotransposon-derived piRNAs known as 'pingpong' has been described 15 . However, pachytene piRNAs, the most abundant class of mammalian piRNAs, are mainly processed from intergenic precursor transcripts devoid of transposons and repetitive elements . The in vitro characterization of a 3′ to 5′ trimming activity for the formation of the 3′ ends of piRNAs has been reported 16 , and although a model for primary piRNA biogenesis has been hypothesized 17 , comprehensive, in vivo evidence is still lacking. Whether pachytene piRNAs target RNAs in trans is also unknown.

In Drosophila melanogaster and many other metazoans, the specification of germ cells requires cytoplasmic inheritance of maternally synthesized RNA and protein determinants, which are assembled in electron-dense cytoplasmic structures known as germ or polar granules, found at the posterior end of the oocytes. Recent studies have shown that the formation of germ granules is dependent on the interaction of proteins containing tudor domains with the piwi-interacting RNA (piRNA)-binding Piwi proteins, and such interactions are dependent on symmetrically dimethylated arginines (sDMAs) of Piwi proteins. Tudor-Piwi interactions are crucial and are conserved in the germ cells of sexually reproducing animals, including mammals. In the September 1, 2010, issue of Genes & Development, Liu and colleagues (pp. 1876-1881) use a combination of genetics, biochemistry, and crystallography to uncover the molecular and structural details of how Tudor recognizes and binds the sDMAs of the Piwi protein Aubergine.

Following the birth in 2018 of two babies from embryos altered using CRISPR-Cas9, human germline gene editing (GGE) moved from abstract concern to reality. He Jiankui, the scientist responsible, has been roundly condemned by most scientific, legal and ethical commentators. However, opinions remain divided on whether GGE could be acceptably used in the future, and how, or if it should be prohibited entirely. The many reviews, summits, positions statements and high-level meetings that have accompanied the emergence of CRISPR technology acknowledge this, calling for greater public engagement to help reach a consensus on how to proceed. These calls are laudable but far from unproblematic. Consensus is not only hugely challenging to reach, but difficult to measure and to know when it might be achieved. Engagement is clearly desirable, but engagement strategies need to avoid the limitations of previous encounters between publics and biotechnology. Here we set CRISPR in the context of the biotechnology and fertility industries to illustrate the lessons to be learned. In particular we demonstrate the importance of avoiding a 'deficit mode' in which resistance is attributed to a lack of public understanding of science, addressing the separation of technical safety criteria from ethical and social matters, and ensuring the scope of the debate includes the political-economic context in which science is conducted and new products and services are brought to market. Through this history, we draw on Mary Douglas' classic anthropological notion of 'matter out of place' to explain why biotechnologies evoke feelings of unease and anxiety, and recommend this as a model for rehabilitating lay apprehension about novel biological technologies as legitimate matters of concern in future engagement exercises about GGE.

Primordial germ cell (PGC) development in the zebra®sh is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in ®sh PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebra®sh, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell-to the 1000-cell stage the protein is highly concentrated in two`dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The ®rst distinct cell-speci®c expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development. q

Background and Aim: Spermatogonial stem cells (SSCs) have previously been isolated from animals' testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. Materials and Methods: The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. s were read and relevant full papers were reviewed for data extraction. Results: From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers repor...

Objective: Identify the etiology of elevated B 12 in autoimmune lymphoproliferative syndrome (ALPS). Design: Peripheral blood of ALPS patients with elevated B 12 and controls were evaluated. Results: Total and holo-haptocorrin (HC) levels were 26-and 23-fold higher in ALPS patients, respectively. No abnormal B 12-binding proteins were found. Western blot revealed HC in lymphocyte lysates only from ALPS patients. Conclusion: Elevated concentrations of B 12 found in ALPS patients were due to increased lymphocyte expression of HC.

Piwi-interacting RNAs (piRNAs) are small RNA molecules of between 24 to 31 nucleotides (nts) sequence and interact with Piwi subfamily proteins. These piRNAs play important regulatory roles in germline stem cell maintenance, epigenetic regulation, and transposition. Although the biogenesis pathways of piRNAs are not well understood but recent scientific advances have revealed some important aspects of their synthesis. Studies on regulatory mechanism of piRNAs are in their early stages. Yet, the current findings suggest that piRNA based regulation has direct implications on germline development and transposon silencing through heterochromatin formation or RNA destabilization. This review summarizes the knowledge about all aspects of piRNAs since its discovery.

Null mutations in the Drosophila gene, slowmo (slmo), result in reduced mobility and lethality in first-instar larvae. Slowmo encodes a mitochondrial protein of unknown function, as do the two other homologs found in Drosophila. Here, we have studied a hypomorphic P-element allele of slmo demonstrating its effects on germline divisions in both testes and ovaries. Using in situ studies, enhancer-trap activity, and promoter fusions, we have shown that slmo expression in testes is found in the somatic cyst cells (SCC). The hypomorphic allele for Slmo revealed apoptotic loss of germline cells in the larval germline, culminating in a complete absence of the germline in adult flies. In females, a similar degeneration of the germarium is observed, while reporter gene expression is found in both germline and somatic cells. Using a null mutation in female germline clones, we find slmo is dispensable from the germline cells. Our results suggest that Slowmo is not required in germline cells directly, but is required in SCCs responsible for maintaining germline survival in both sexes. genesis 45:66–75, 2007. © 2007 Wiley-Liss, Inc.

MicroRNAs guide many aspects of development in all metazoan species. Frequently, microRNAs are expressed during a specific developmental stage to perform a temporally defined function. The C. elegans mir-35-42 microRNAs are expressed abundantly in oocytes and early embryos and are essential for embryonic development. Here, we show that these embryonic microRNAs surprisingly also function to control the number of progeny produced by adult hermaphrodites. Using a temperature-sensitive mir-35-42 family mutant (a deletion of the mir-35-41 cluster), we demonstrate three distinct defects in hermaphrodite fecundity. At permissive temperatures, a mild sperm defect partially reduces hermaphrodite fecundity. At restrictive temperatures, somatic gonad dysfunction combined with a severe sperm defect sharply reduces fecundity. Multiple lines of evidence, including a late embryonic temperature-sensitive period, support a role for mir-35-41 early during development to promote subsequent sperm production in later larval stages. We further show that the predicted mir-35 family target sup-26 (suppressor-26) acts downstream of mir-35-41 in this process, suggesting that sup-26 de-repression in mir-35-41 deletion mutants may contribute to temperature-sensitive loss of fecundity. In addition, these microRNAs play a role in male fertility, promoting proper morphogenesis of male-specific mating structures. Overall, our results demonstrate that robust activity of the mir-35-42 family microRNAs not only is essential for embryonic development across a range of temperatures but also enables the worm to subsequently develop full reproductive capacity. KEYWORDS fertility maternal effect germline sperm male fertility microRNAs are a class of endogenous 22-23-nucleotide RNAs that repress expression of complementary target mRNAs to govern diverse developmental and physiological processes in essentially all complex eukaryotes. In most cases, mature microRNAs are generated from much longer transcripts through a series of nucleolytic cleavages and subsequently loaded into complexes with Argonaute proteins (Ketting 2011). Together with the effector protein GW182, the microRNAloaded Argonaute forms the RNA-induced silencing complex (miRISC), which inhibits the translation and/or stability of complementary target mRNAs. The seed region (nucleotides 2-7) of a microRNA is the most important for determining target specificity (Bartel 2009). microRNAs that share the same seed sequence are classified as a "family" because they can potentially bind and redundantly regulate the same set of target mRNAs.

IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytrzcha ,fallax and 0. rrifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT. . .AATG, and thereby resembles a TBEl transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This imulies a excision mechanism for IES-LA that is distinct from those for other known Oxvtricha IESs.

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud(1) mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.

mAlthough human and mouse antibodies are similar when one considers their diversification strategies, they differ in the extent to which kappa and lambda light chains are present in their respective variable light chain repertoires. While the Igk-V germline genes are preponderant in mice (95% or more), they comprise only 60% in humans. This may account for differences in the structural repertoire encoded in the Igk-V germline genes of these species. However, this subject has not been properly investigated, partially because a systematic structural characterization of the mouse Igk-V germline genes has not been undertaken. In the present study we compiled all available information on mouse Igk-V germline genes to characterize their structural repertoire. As expected, comparison with the structural repertoire of human Igk-V germline genes indicates differences. The most interesting is that the mouse Igk-V germline gene repertoire is more diverse in structural terms than its human counterpart: the mouse encodes seven canonical structure classes (combination of canonical structures in L1 and L3). In contrast, the human encodes only four. Analysis of the evolutionary relationships of human and mouse Igk-V germline genes led us to propose that the difference reflects a strategy of mice to compensate for the small lambda chain contribution to the repertoire of their variable light chains.

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.

Associations of sarcoma with inherited cancer syndromes implicate genetic predisposition in sarcoma development. However, due to the apparently sporadic nature of sarcomas, little attention has been paid to the role genetic susceptibility in sporadic sarcoma. To address this, we performed targetedgenomic sequencing to investigate the prevalence of germline mutations in known cancer-associated genes within an Asian cohort of sporadic sarcoma patients younger than 50 years old. We observed 13.6% (n = 9) amongst 66 patients harbour at least one predicted pathogenic germline mutation in 10 cancer-associated genes including ATM, BRCA2, ERCC4, FANCC, FANCE, FANCI, MSH6, POLE, SDHA and TP53. The most frequently affected genes are involved in the DNA damage repair pathway, with a germline mutation prevalence of 10.6%. Our findings suggests that genetic predisposition plays a larger role than expected in our Asian cohort of sporadic sarcoma, therefore clinicians should be aware of the possibility that young sarcoma patients may be carriers of inherited mutations in cancer genes and should be considered for genetic testing, regardless of family history. The prevalence of germline mutations in DNA damage repair genes imply that therapeutic strategies exploiting the vulnerabilities resulting from impaired DNA repair may be promising areas for translational research. Sarcoma comprises a family of mesenchymal tumors that arise predominantly in the bone or soft tissue. They are heterogeneous with over 70 histological subtypes 1, 2 , develop from various anatomical sites and can affect patients across age groups 3. Sarcomas account for 1% of adult solid tumors and nearly 21% of pediatric solid tumors, and carry a poor prognosis, primarily due to late diagnosis and advanced disease at presentation 3. Although mostly sporadic in occurrence, sarcoma has also been associated with hereditary cancer syndromes 2, 4. A primary example is Li-Fraumeni Syndrome (LFS), an autosomal-dominant cancer syndrome associated with germline

Housekeeping genes are often used as references when quantifying the relative abundance of transcripts of interest, because it is assumed that they are stably expressed across tissues and developmental stages. Standard housekeeping genes are targeted particularly in organisms where there is no detailed information on gene expression profiles. Here, the validity of using the two widely accepted housekeeping genes, 18S rRNA and ␤-actin, as reference genes to normalize real-time RT-PCR gene expression data from the Kuruma shrimp, Marsupenaeus japonicus, was tested. Expression patterns of two target genes in a diverse sample set of embryonic, larval, post-larval and gonad mRNAs were quantified using relative and absolute real-time RT-PCR procedures. Comparison of these approaches revealed significant differences (P < 0.0001) in transcript level profiles between the relative and absolute procedures for both target genes. When 18S rRNA was used as a reference, target gene expression was more similar to that of the absolute method than when ␤-actin was used as a reference. Variability between the relative and absolute procedures occurred for a greater percentage of the embryonic stages compared to later developmental stages. This study indicates that the use of 18S rRNA and ␤-actin for studying relative gene expression patterns in Kuruma shrimp embryonic, larval, post-larval and gonad samples will give significantly variable results, and illustrates the proposition that housekeeping genes are not necessarily appropriate references for real-time RT-PCR data normalization. Until suitable reference genes are characterized, gene expression experiments using the studied Kuruma shrimp tissues of different morphological developmental stages should use absolute quantification procedures.

The nanos-class gene of the leech Helobdella robusta (Hro-nos) is present as a maternal transcript whose levels decay during cleavage; HRO-NOS protein is more abundant in the D quadrant cells relative to the A, B, and C quadrants; and HRO-NOS is more abundant in the ectodermal precursor cell (DNOPQ) than in its sister mesodermal precursor (DM) (Pilon and Weisblat, 1997). Here, using in situ hybridization, we show that Hro-nos mRNA is broadly distributed throughout the zygote, is concentrated in both animal and vegetal teloplasm during stage 1 and is at higher levels in DNOPQ than in DM at stage 4b. Hro-nos expression increases after stage 7, as judged by in situ hybridization, developmental RT-PCR, and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9 and 10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive "spots" comprising of one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin, arising in a segmentally iterated manner from the M lineage, and correspond to cells previously proposed as primordial germ cells (PGCs; Bü rger, 1891; Weisblat and Shankland, 1985). These results support the proposal that nanos-class genes functioned in the specification of germline cells in the ancestral bilaterian and possibly in a separate process related to embryonic polarity in the ancestral protostome.

The products of the maternal-effect genes, nanos (nos) and oskar (osk), are important for the development of germ cells in insects. Furthermore, these genes have been proposed as candidates for donating functional DNA regulatory sequences for use in gene drive systems to control transmission of mosquito-borne pathogens. The nos and osk genes of the cosmopolitan vector mosquito, Culex quinquefasciatus, encode proteins with domains common to orthologues found in other mosquitoes. Expression analyses support the conclusion that the role of these genes is conserved generally among members of the nematocera. Hybridization in situ analyses reveal differences in mRNA distribution in early embryos in comparison with the cyclorraphan, Drosophila melanogaster, highlighting a possible feature in the divergence of the clades each insect represents.

Antibodies are critical entities used for the protection of an individual organism against infection and disease. Through a complex series of events genes rearrange to encode repertoires of molecules, which are tested for their ability to identify foreign molecules. In this report, we discuss factors defined by the individual as well as by infectious organisms that shape the antibody repertoire. A more thorough understanding of the interplay between these factors will eventually allow us to elucidate the underlying mechanisms of disease susceptibility following infection, and to design potent vaccines and other immunomodulating reagents.

Housekeeping genes are often used as references when quantifying the relative abundance of transcripts of interest, because it is assumed that they are stably expressed across tissues and developmental stages. Standard housekeeping genes are targeted particularly in organisms where there is no detailed information on gene expression profiles. Here, the validity of using the two widely accepted housekeeping genes, 18S rRNA and ␤-actin, as reference genes to normalize real-time RT-PCR gene expression data from the Kuruma shrimp, Marsupenaeus japonicus, was tested. Expression patterns of two target genes in a diverse sample set of embryonic, larval, post-larval and gonad mRNAs were quantified using relative and absolute real-time RT-PCR procedures. Comparison of these approaches revealed significant differences (P < 0.0001) in transcript level profiles between the relative and absolute procedures for both target genes. When 18S rRNA was used as a reference, target gene expression was more similar to that of the absolute method than when ␤-actin was used as a reference. Variability between the relative and absolute procedures occurred for a greater percentage of the embryonic stages compared to later developmental stages. This study indicates that the use of 18S rRNA and ␤-actin for studying relative gene expression patterns in Kuruma shrimp embryonic, larval, post-larval and gonad samples will give significantly variable results, and illustrates the proposition that housekeeping genes are not necessarily appropriate references for real-time RT-PCR data normalization. Until suitable reference genes are characterized, gene expression experiments using the studied Kuruma shrimp tissues of different morphological developmental stages should use absolute quantification procedures.

Aging studies on diverse species ranging from yeast to man have culminated in the delineation of several signaling pathways that influence the process of senescent decline and aging. While understanding these interlinked signal-transduction cascades is becoming even more detailed and comprehensive, the cellular and biochemical processes they impinge upon to modulate the rate of senescent decline and aging have lagged considerably behind. This fundamental question is one of the most important challenges of modern aging research and has been the focus of recent research efforts. Emerging findings provide insight into the facets of cellular metabolism which can be fine-tuned by upstream signaling events to ultimately promote longevity. Here, we survey the mechanisms regulating aging in the simple nematode worm Caenorhabditis elegans, aiming to highlight recent discoveries that shed light into the interface between aging signaling pathways and cellular energy metabolism. Our objective is to review the current understanding of the processes involved and discuss mechanisms that are likely conserved in higher organisms.

The O-antigen of the gram negative bacteria Brucella is composed of an homopolymer of 4,6-dideoxy-4-formamido-␣-d-mannopyranosyl (or perosamine). Several mAb interact specifically with only the O-antigen of certain Brucella species. Although, many studies show that this specific recognition results mainly from the ratios of ␣1-2 and ␣1-3 link between the different Brucella strain perosamine residues, little is known about the mAb recognising this O-antigen. In this paper, we describe the binding profile of five anti-Brucella O-antigen mAb to the LPS of two Brucella strains and a bacteria possessing a nearly identical O-antigen: Yersinia enterocolitica O:9. We show that the specificity of these five mAb can be correlated to their germ line gene usage. Besides, their relative affinity to the different LPS is correlated to their ability to protect against Brucella infection by passive transfer in a mouse model. The analysis of their 3D structure gives new hypothesis of the epitopes recognised.

Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol(1) mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.

The human genome contains frequent single-basepair variants that may or may not cause genetic disease. To characterize benign vs. pathogenic missense variants, numerous computational algorithms have been developed based on comparative sequence and/or protein structure analysis. We compared computational methods that use evolutionary conservation alone, amino acid (AA) change alone, and a combination of conservation and AA change in predicting the consequences of 254 missense variants in the CDKN2A (n 5 92), MLH1 (n 5 28), MSH2 (n 5 14), MECP2 (n 5 30), and tyrosinase (TYR) (n 5 90) genes. Variants were validated as either neutral or deleterious by curated locus-specific mutation databases and published functional data. All methods that use evolutionary sequence analysis have comparable overall prediction accuracy (72.9-82.0%). Mutations at codons where the AA is absolutely conserved over a sufficient evolutionary distance (about one-third of variants) had a 91.6 to 96.8% likelihood of being deleterious. Three algorithms (SIFT, PolyPhen, and A-GVGD) that differentiate one variant from another at a given codon did not significantly improve predictive value over conservation score alone using the BLOSUM62 matrix. However, when all four methods were in agreement (62.7% of variants), predictive value improved to 88.1%. These results confirm a high predictive value for methods that use evolutionary sequence conservation, with or without considering protein structural change, to predict the clinical consequences of missense variants. The methods can be generalized across genes that cause different types of genetic disease. The results support the clinical use of computational methods as one tool to help interpret missense variants in genes associated with human genetic disease. Hum Mutat 28 , 683-693, 2007. Published 2007

We recently completed a map of the human immunoglobulin lambda (IGL) 1 MRC Centre for Protein locus on chromosome 22q11.2 and showed that the V l genes are arranged Engineering, Hills Road Cambridge, CB2 2QH, UK in three distinct clusters, each containing members of different V l families.