BACKGROUND
many aspects of the nematode caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model.
because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as rnai screens, can be rapidly tested.
this is an obvious advantage when compared to the increasingly complex drosophila and mouse genomes.
therefore, the nematode c. elegans has emerged as an excellent entry point to begin to address these predictions.
there are currently two main approaches in c. elegans to investigate gene function on a genomic scale using reverse-genetics.
the first one is based on rna-mediated interference  where "functional knock-outs" of a particular gene can be studied and phenotypes identified.
the other is a pcr -based technology to identify genetic mutants in a mutagenized library.
complementary expression data can also be acquired from microarray data or by serial analysis of gene expression  to identify genes that are co-expressed or up/down regulated under defined conditions.
these methods can be directly combined, on a particular set of genes, to provide a comprehensive description of the gene's expression patterns and functions  <cit> .
in addition, results can be refined using independent screens of other experimental systems, for example human cell lines.
rnai based on the introduction of double-stranded rna  is the method that results in specific inactivation of the corresponding gene through the degradation of endogenous mrna.
it was originally described in c. elegans  <cit>  and has become the main reverse-genetics tool for determining the function of specific genes.
several large-scale studies involving c. elegans  <cit> , subjected approximately 90% of the  <dig>  predicted genes to down regulation via rnai.
moreover, individual clones of the entire rnai feeding library described in fraser et al.
<cit>  can be ordered directly.
in addition, in vitro synthesis of ~21-nt small interfering rnas to mediate gene-specific suppression in mammalian cells have been developed in order to extend this particular technique to higher eukaryotes  <cit> .
80% of c. elegans genes have human homologs  <cit> .
as long as we are able to establish orthology, information obtained for a gene sequence in one organism is potentially transferable to the other  <cit> .
nevertheless, since the human and the worm genomes are phylogenetically very distant, in many cases these sequences are only able to produce poorly supported trees.
moreover, position of individual branches may not be conserved between different algorithms for finding distance trees.
phylogenetic reconstructions involving human/worm sequences in a number of cases are unable to resolve their exact phylogenetic past.
in the case of orthology assignments they provide only an indication for its existence.
moreover, sequence orthology does not necessarily imply the same function.
however, genes shown to be descendants of the same gene  have, in general retained the same function over the course of evolution  <cit> .
if so, it is important to select for analysis only the genes where a clear orthology assignment can be established.
based on a collection of uncharacterized protein families derived from a comparison of three available genomes including h. sapiens, c. elegans and d. melanogaster, an integrated in-house program for functional gene annotation was initiated.
as part of this annotation effort, a set of genes originating from this collection of novel protein families was selected for functional analysis in both c. elegans and human cell lines.
in this study novel protein families are explored using a diverse set of technologies involving both in silico and experimental analysis with the intention of identifying interesting gene candidates showing evidence of highly conserved function which may serve as potential drug targets in the future.
we have sought to identify, through phylogenetic analysis, the most likely worm orthologs for a set of human genes.
expression patterns and rnai data in the worm were obtained, as well as corresponding data using human cell lines.
RESULTS
phylogenetic analysis: the most likely ortholog?
multiple gene duplication followed by massive gene loss and acquisition of novel functions has been shaping the evolution of distant organisms.
in a search for the most likely orthologs, special attention must be paid to the fate of duplicated genes when related to speciation.
duplicated genes tend to evolve in different patterns following the duplication event arising from different functional constrains  <cit> .
according to remm et al.
and sonnhammer and koonin, out-paralogs are paralogs that predate speciation  <cit> .
by contrast, in-paralogs are genes that arose after speciation.
therefore, all in-paralogs are considered potential functional orthologs.
using translated sequence we compare maximum parsimony trees to neighbor joining counterparts.
genes for which the position of the branches  was conserved between both algorithms chosen for finding distance trees , were selected for the experimental study .
this criterion has been used in order to eliminate genes producing unstable topologies caused by duplications or a generally weak phylogenetic signal.
also, in this way it was possible to infer the homolog which most likely retained the ancestral gene function.
unique orthologous gene pairs are difficult to identify using standard similarity searches, as multiple candidate genes are typically obtained.
our trees typically contain two or more human homologs showing significant sequence similarity to a single worm gene .
therefore, we selected candidate genes by choosing genes that produced trees that allow for identification of single human sequences as the most probable orthologs.
these trees clearly discriminate out-paralogs or other in-paralogs .
in effect, well-supported trees showing a one-to-one relationship between human and worm sequence were selected.
however, since the purpose of this analysis was to identify genes that most likely retained the same function rather than orthology sensu stricto, it is possible that in some cases a paralog  was selected as the most promising ortholog.
in other words, the c. elegans genes that we identified are either true orthologs or the best in-paralogs to the corresponding human genes  <cit> .
despite the fact that the exact type of orthology encountered in individual trees could not always be identified, we find this level of resolution satisfactory for the purposes of this study.
thus, phylogenetic analysis  predicts f41d <dig>   to have a similar function to human caa <dig>  the tree also assigns other human sequences, namely baa <dig> and aal <dig>  which are apparently a product of a duplication event in the mouse/human lineages, as either out-paralogs or in-paralogs.
regardless of their exact orthology/paralogy relation to the worm sequence, neither of them is the most probable ortholog.
to the contrary, a frequently encountered situation was obtained in the case of the worm gene c13f <dig>  .
the two human sequences though rather similar having a protein matrix distance of  <dig>  , were easily separated in both the neighbor joining tree  and the maximum parsimony tree .
the nj tree in particular demonstrates that the gene was duplicated in the animal series starting from gallus sp.
and upwards.
thus, in an ideal case, phylogenetic analyzes would correctly identify the exact copy of the duplicated gene in the human genome and assign it to a single c. elegans sequence as its true ortholog, as shown for f41d <dig>  .
the position of the other copy would point to its paralogous/in-paralogous relation to the worm sequence.
however, the worm gene is either weakly associated with the branch leading to both human sequences  and it forms a separate branch with the xp_ <dig> human sequence .
c. elegans genes and their most probable human orthologs are shown in the tab.
<dig>  interestingly, due to the strict criteria we applied in order to select the most likely human orthologs, even c27a <dig> , a known disease gene , which is regarded as orthologous to the human gene nppase  was not identified.
expression profiles and rnai phenotypes
a distinct gfp expression: f41d <dig> , c17e <dig> , zk <dig>  and c09d <dig>
here we report gfp expression under the control of a putative promoter for f41d <dig>  that occurs predominantly in many neural cells in the head around the posterior pharyngeal bulb, along the ventral nerve cord and in the tail .
head neurons include clusters in the dorsal and retrovesicular ganglia as well as the sensory amphid neurons.
expression is present in both larval and adult stages.
no other phenotype was observed following down-regulation which is in accordance to previously published results by  <cit> .
the c17e <dig> ::gfp reporter is expressed in the developing embryo and l <dig> larval stages, in three distinct sheath/socket cells in the head region close to the anterior bulb of the pharynx and several cells around the anus .
expression is also seen in several pharyngeal muscle cells.
dye filling tests confirmed that the processes extending to the nose were not from amphid neurons, but rather from socket cells .
the expression occurs exclusively in the developing embryo and l <dig> larval stage suggesting involvement of the gene in development.
no rnai phenotype was associated with this gene.
the wild type phenotype, following down-regulation, was also obtained by simmer et al.
<cit> .
however, the results obtained by piano et al.
<cit>  show that silencing of the gene results in embryonic lethality.
nevertheless, inconsistent phenotypes have been obtained by different groups for several other genes  <cit> .
zk <dig> ::gfp expression pattern includes spermatheca, hypodermal cells, pharynx and the excretory cell and channels .
in the l <dig> stage, expression was seen in the vulva, and in p <dig> p descendants.
c09d <dig>  had a simple expression pattern limited to intestinal cells in all developmental stages .
visible rnai phenotypes: t24d <dig> , f23c <dig> , zk <dig>  and b <dig> a
to investigate in detail the rnai effects on growth, brood size and life span, we have closely followed a population of embryos  to full development at 20°c, in rnai plates for corresponding genes with visible rnai phenotypes.
c. elegans sqv- <dig>  <dig> protein  encoded by t24d.
<dig> gene is a chondroitin synthase that initiates/elongates chondroitin chains.
this protein is also required for cytokinesis, gonad migration and vulval morphogenesis where it possibly promotes filling an extracellular space with fluid  <cit> .
following exposure to the corresponding dsrna, a smaller f <dig> population size was observed when compared to the control animals in the both n <dig> and nl <dig>  f <dig> animals show a robust rnai-induced phenotype including: sickly appearance, partial sterility, and small brood size.
lethality was observed in f <dig> embryos in the rrf- <dig> background at 20°c.
the partial sterility among the f <dig> animals is in accordance with the reported  mutant phenotype .
all the phenotypes indicated above were reduced in the n <dig> background and also when grown at 15°c.
additional mutant phenotypes included squashed vulva , and reduced l <dig> vulva invagination .
however, according to fraser et al.
<cit> , down-regulation via rnai did not produce a distinct phenotype.
this is possibly due to the large-scale nature of their study and its focus on early developmental stages.
for f23c <dig> , our results revealed slow growth  and uncoordinated behavior  which are both in agreement with the results obtained by fraser et al.
<cit> .
our observations also confirmed larval arrest as reported previously  <cit> .
as with t24d.
<dig>  zk <dig>  and b <dig> a, down-regulation of f23c <dig>  did not affect life span.
gene function related to growth, larval development and locomotory behavior have been also inferred from the mutant phenotype .
down-regulation of zk <dig>  affects the growth rate significantly in both wild type and the hypersensitive nl <dig> strain .
at 20°c growth was 10– <dig> times slower then the wild type control, slightly more pronounced in the rrf- <dig> strain.
a large proportion of sterile f <dig>  animals was also observed.
the minority of animals that were fertile produced very few eggs but these eggs were as viable as wild type.
according to the previous studies down-regulation of the same gene has produced the following phenotypes: gro, emb, stp, lva  <cit> .
our results for down-regulation of b <dig> a are consistent with the data obtained by kamath et al.
<cit> .
rnai resulted in an egl phenotype that was more pronounced in the rrf- <dig> background.
the involvement of the gene in oviposition was inferred from the mutant phenotype .
the gene has orthologous sequences in both the human and mouse genomes that code for a small ctd phosphatase and nuclear lim interactor-interacting factor  <dig>  respectively.
rnai on c05c <dig>  has been reported by simmer et al.
as emb, lva and lvl  <cit> .
however, our results are consistent with another study published by kamath et al.
where no specific phenotype was observed  <cit> .
the mutant phenotype of c05c <dig>  points to its involvement in embryonic and larval development with a molecular function associated with protein binding .
although a greater proportion of genes show specific rnai phenotypes when using rrf- <dig> strain  <cit> , our results point to an increase in the severity of the phenotypes following down-regulation in the rrf- <dig> background, rather than additional phenotypes.
subcellular localization in human cell lines
for seven of the human orthologs presented in this study, we cloned full-length open reading frames , representing encoded cdnas, into vectors containing v <dig> epitope sequences in order to generate recombinant fusion proteins for immunofluorescence detection.
because of the novelty of these proteins and the lack of available antibodies against such proteins, it was necessary to overexpress in human cell lines and subsequently detect with antibodies against the v <dig> fusion tag.
subcellular localizations were obtained in two human cell lines: hela and human embryonic kidney  <dig> .
endogenous expression for the proteins studied in the two cell lines was confirmed by rt-pcr.
np_ <dig>  human ortholog for zk <dig> , displays three distinct patterns: diffuse nuclear, nucleolar and nuclear foci .
we were able to confirm the nucleolar pattern by colocalization with fibrillarin, a protein predominantly found in nucleoli and cajal bodies.
we were unable to determine the exact sub-compartment to which the nuclear foci belong.
however, the pattern is strikingly similar to that of paraspeckles.
the proteins, described as u <dig> snornp protein  <dig> homologs, belong to the imp <dig> family of proteins which are small ribonucleoproteins involved in pre-ribosomal rna processing.
these results are consistent with those previously obtained in yeast and recently described in human  <cit> .
based on the interaction of these proteins with snorna in 60-80s rnp complexes and their likely involvement in pre-rrna processing, it is possible to speculate that the preferred location of the imp <dig> protein is both transient and dynamic throughout the nucleus, which would explain the three patterns we observe.
np_ <dig> also described as hypothetical protein mgc <dig> and predicted ortholog of worm protein c17e <dig>  displayed a nuclear and perinuclear pattern covering the entire circumference of the nucleus but did not appear to be inside the nucleus.
we were able to confirm the presence of this protein at the nuclear membrane by colocalization with lamin, denoting a possible role for this protein as part of the nuclear envelope .
c3hc <dig> which contains a ring-finger motif, has been classified as the third member  of the recently defined membrane-associated ring-ch protein family  <cit> .
the same authors reported that proteins belonging to this family, including march-iii, characteristically contain two predicted c-terminal transmembrane domains, indicating a possible association with membrane-bound organelles.
furthermore, bartee et al.
concluded that localization of human march iii by fusion protein overexpression revealed a punctate pattern partially overlapping with cytoplasmic vesicles, specifically early endosomes.
however, we were unable to confirm this finding.
it is important to note that proteins are, in many cases, dynamic in location meaning that there are potentially multiple locations.
thus, our co-staining data are the most compelling we have seen.
aak25825/np_ <dig>  also described as btb/poz domain containing  <dig> , exhibited a cytoplasmic pattern resembling elongated "worm-like" bodies for which we were unable to determine a known structure .
members of this domain family have been shown to interact with co-repressor complexes involved in transcriptional repression  <cit> .
previously, btbd <dig> has been shown to interact specifically with topoisomerase i  <cit> .
in addition, a recent study further characterized btbd <dig> as colocalizing with trim family members  <cit> .
interestingly, trim proteins have been shown to exhibit ubiquitin ligase activity.
np_ <dig> has no functional annotation and has only been described as an open reading frame located on human chromosome  <dig>  this protein exhibits weak expression when "overexpressed" under control of cmv promoter.
immunofluourescence shows this protein to have both a cytoplasmic and a nuclear distribution.
furthermore, the cytoplasmic expression appears punctate and partially colocalizes with calreticulin, a marker for endoplasmic reticulum .
aap <dig> /jc <dig> is a member of a family of small c-terminal domain phosphatase  and contains an nif domain .
other members of this family have been shown to interact with rna polymerase ii and show nuclear localization   <cit> .
surprisingly, scp <dig> was detected at the plasma membrane by colocalization with annexin ii, a known component of the plasma membrane .
in addition to this pattern, we noted fibrous structures that partially colocalized with beta-actin .
hypothetical protein np_ <dig> expression was detected by western blot and immunofluorescence microscopy .
microscopy detection displayed a cytoplasmic/nuclear rim staining pattern.
however, western blot analysis revealed a molecular weight of approximately  <dig> kda which is inconsistent with the predicted size of  <dig> kda, indicating a possible premature stop in translation or post-translational cleavage.
in addition, we were unable to detect ectopic expression for np_ <dig>  any attempts to detect this protein via western blot or microscopy were unsuccessful despite sequence validation of the corresponding expression plasmid.
CONCLUSIONS
specific expression patterns have been identified for the f41d <dig> , zk <dig> , c17e <dig>  and c09d <dig>  gfp-fusion strains.
visible rnai phenotypes for t24d <dig> , f23c <dig> , zk <dig>  and b <dig> a were observed.
rnai on t24d <dig> a produced an extensive phenotype that was not revealed in the large-scale study by fraser et al.
<cit> .
thus, our study shows the value of analyzes focusing on a small number of candidate genes where a variety rather then a pre-defined set of phenotypes are observed.
in addition, immunofluorescence microscopy of human cell lines over-expressing np_ <dig>  np_ <dig>  aak25825/np_ <dig>  np_ <dig>  aap <dig> /jc <dig> and np_ <dig> has detected subcellular localization of the corresponding proteins.
the most complete data set comprising both a visible rnai phenotype or/and a distinct gfp expression in c. elegans and a subcellular protein over-expression profile using human cell lines has been obtained for zk <dig>  , f41d <dig>   and c17e <dig>   .
in the case of genes f41d <dig>  and c17e <dig> , a mainly neuronal gfp expression was detected.
therefore, the absence of an observable rnai phenotype may be explained by the known refractory nature of rnai in this tissue.
for the gene zk <dig> , which showed a more generalized effect on the development, corresponding widespread expression pattern was observed in the worm and in human cell lines.
given that the protein prediction indicates this gene as a snornp candidate, its involvement in a variety of cellular processes thus can be expected.
to the contrary, restricted subcellular expression patterns in human cells observed in a few cases, for example the aak <dig>  and np_115683c11d <dig>  genes , correlate with the absence of phenotypes other than the wild type.
this fact suggests that these genes may be of somewhat lesser importance.
in conclusion, this comparative c. elegans-human cell lines study based on orthology assignments explores gene functionality by combining two key aspects of functional genomics: a whole organism approach and protein overexpression data using cells lines.
our results extend currently available information on the selected genes providing a step more towards identifying their exact function.

 many aspects of the nematode caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as rnai screens, can be rapidly tested. this is an obvious advantage when compared to the increasingly complex drosophila and mouse genomes. therefore, the nematode c. elegans has emerged as an excellent entry point to begin to address these predictions. there are currently two main approaches in c. elegans to investigate gene function on a genomic scale using reverse-genetics. the first one is based on rna-mediated interference where "functional knock-outs" of a particular gene can be studied and phenotypes identified. the other is a pcr -based technology to identify genetic mutants in a mutagenized library. complementary expression data can also be acquired from microarray data or by serial analysis of gene expression to identify genes that are co-expressed or up/down regulated under defined conditions. these methods can be directly combined, on a particular set of genes, to provide a comprehensive description of the gene's expression patterns and functions. in addition, results can be refined using independent screens of other experimental systems, for example human cell lines. rnai based on the introduction of double-stranded rna is the method that results in specific inactivation of the corresponding gene through the degradation of endogenous mrna. it was originally described in c. elegans and has become the main reverse-genetics tool for determining the function of specific genes. several large-scale studies involving c. elegans, subjected approximately 90% of the predicted genes to down regulation via rnai. moreover, individual clones of the entire rnai feeding library described in fraser et al. can be ordered directly. in addition, in vitro synthesis of ~21-nt small interfering rnas to mediate gene-specific suppression in mammalian cells have been developed in order to extend this particular technique to higher eukaryotes. 80% of c. elegans genes have human homologs. as long as we are able to establish orthology, information obtained for a gene sequence in one organism is potentially transferable to the other. nevertheless, since the human and the worm genomes are phylogenetically very distant, in many cases these sequences are only able to produce poorly supported trees. moreover, position of individual branches may not be conserved between different algorithms for finding distance trees. phylogenetic reconstructions involving human/worm sequences in a number of cases are unable to resolve their exact phylogenetic past. in the case of orthology assignments they provide only an indication for its existence. moreover, sequence orthology does not necessarily imply the same function. however, genes shown to be descendants of the same gene have, in general retained the same function over the course of evolution. if so, it is important to select for analysis only the genes where a clear orthology assignment can be established. based on a collection of uncharacterized protein families derived from a comparison of three available genomes including h. sapiens, c. elegans and d. melanogaster, an integrated in-house program for functional gene annotation was initiated. as part of this annotation effort, a set of genes originating from this collection of novel protein families was selected for functional analysis in both c. elegans and human cell lines. in this study novel protein families are explored using a diverse set of technologies involving both in silico and experimental analysis with the intention of identifying interesting gene candidates showing evidence of highly conserved function which may serve as potential drug targets in the future. we have sought to identify, through phylogenetic analysis, the most likely worm orthologs for a set of human genes. expression patterns and rnai data in the worm were obtained, as well as corresponding data using human cell lines.  phylogenetic analysis: the most likely ortholog? multiple gene duplication followed by massive gene loss and acquisition of novel functions has been shaping the evolution of distant organisms. in a search for the most likely orthologs, special attention must be paid to the fate of duplicated genes when related to speciation. duplicated genes tend to evolve in different patterns following the duplication event arising from different functional constrains. according to remm et al. and sonnhammer and koonin, out-paralogs are paralogs that predate speciation. by contrast, in-paralogs are genes that arose after speciation. therefore, all in-paralogs are considered potential functional orthologs. using translated sequence we compare maximum parsimony trees to neighbor joining counterparts. genes for which the position of the branches was conserved between both algorithms chosen for finding distance trees, were selected for the experimental study. this criterion has been used in order to eliminate genes producing unstable topologies caused by duplications or a generally weak phylogenetic signal. also, in this way it was possible to infer the homolog which most likely retained the ancestral gene function. unique orthologous gene pairs are difficult to identify using standard similarity searches, as multiple candidate genes are typically obtained. our trees typically contain two or more human homologs showing significant sequence similarity to a single worm gene. therefore, we selected candidate genes by choosing genes that produced trees that allow for identification of single human sequences as the most probable orthologs. these trees clearly discriminate out-paralogs or other in-paralogs. in effect, well-supported trees showing a one-to-one relationship between human and worm sequence were selected. however, since the purpose of this analysis was to identify genes that most likely retained the same function rather than orthology sensu stricto, it is possible that in some cases a paralog was selected as the most promising ortholog. in other words, the c. elegans genes that we identified are either true orthologs or the best in-paralogs to the corresponding human genes. despite the fact that the exact type of orthology encountered in individual trees could not always be identified, we find this level of resolution satisfactory for the purposes of this study. thus, phylogenetic analysis predicts f41d to have a similar function to human caa the tree also assigns other human sequences, namely baa and aal which are apparently a product of a duplication event in the mouse/human lineages, as either out-paralogs or in-paralogs. regardless of their exact orthology/paralogy relation to the worm sequence, neither of them is the most probable ortholog. to the contrary, a frequently encountered situation was obtained in the case of the worm gene c13f. the two human sequences though rather similar having a protein matrix distance of, were easily separated in both the neighbor joining tree and the maximum parsimony tree. the nj tree in particular demonstrates that the gene was duplicated in the animal series starting from gallus sp. and upwards. thus, in an ideal case, phylogenetic analyzes would correctly identify the exact copy of the duplicated gene in the human genome and assign it to a single c. elegans sequence as its true ortholog, as shown for f41d. the position of the other copy would point to its paralogous/in-paralogous relation to the worm sequence. however, the worm gene is either weakly associated with the branch leading to both human sequences and it forms a separate branch with the xp_ human sequence. c. elegans genes and their most probable human orthologs are shown in the tab. interestingly, due to the strict criteria we applied in order to select the most likely human orthologs, even c27a, a known disease gene, which is regarded as orthologous to the human gene nppase was not identified. expression profiles and rnai phenotypes a distinct gfp expression: f41d, c17e, zk and c09d here we report gfp expression under the control of a putative promoter for f41d that occurs predominantly in many neural cells in the head around the posterior pharyngeal bulb, along the ventral nerve cord and in the tail. head neurons include clusters in the dorsal and retrovesicular ganglia as well as the sensory amphid neurons. expression is present in both larval and adult stages. no other phenotype was observed following down-regulation which is in accordance to previously published results by. the c17e::gfp reporter is expressed in the developing embryo and l larval stages, in three distinct sheath/socket cells in the head region close to the anterior bulb of the pharynx and several cells around the anus. expression is also seen in several pharyngeal muscle cells. dye filling tests confirmed that the processes extending to the nose were not from amphid neurons, but rather from socket cells. the expression occurs exclusively in the developing embryo and l larval stage suggesting involvement of the gene in development. no rnai phenotype was associated with this gene. the wild type phenotype, following down-regulation, was also obtained by simmer et al.. however, the results obtained by piano et al. show that silencing of the gene results in embryonic lethality. nevertheless, inconsistent phenotypes have been obtained by different groups for several other genes. zk::gfp expression pattern includes spermatheca, hypodermal cells, pharynx and the excretory cell and channels. in the l stage, expression was seen in the vulva, and in p p descendants. c09d had a simple expression pattern limited to intestinal cells in all developmental stages. visible rnai phenotypes: t24d, f23c, zk and b a to investigate in detail the rnai effects on growth, brood size and life span, we have closely followed a population of embryos to full development at 20°c, in rnai plates for corresponding genes with visible rnai phenotypes. c. elegans sqv- protein encoded by t24d. gene is a chondroitin synthase that initiates/elongates chondroitin chains. this protein is also required for cytokinesis, gonad migration and vulval morphogenesis where it possibly promotes filling an extracellular space with fluid. following exposure to the corresponding dsrna, a smaller f population size was observed when compared to the control animals in the both n and nl f animals show a robust rnai-induced phenotype including: sickly appearance, partial sterility, and small brood size. lethality was observed in f embryos in the rrf- background at 20°c. the partial sterility among the f animals is in accordance with the reported mutant phenotype. all the phenotypes indicated above were reduced in the n background and also when grown at 15°c. additional mutant phenotypes included squashed vulva, and reduced l vulva invagination. however, according to fraser et al., down-regulation via rnai did not produce a distinct phenotype. this is possibly due to the large-scale nature of their study and its focus on early developmental stages. for f23c, our results revealed slow growth and uncoordinated behavior which are both in agreement with the results obtained by fraser et al.. our observations also confirmed larval arrest as reported previously. as with t24d. zk and b a, down-regulation of f23c did not affect life span. gene function related to growth, larval development and locomotory behavior have been also inferred from the mutant phenotype. down-regulation of zk affects the growth rate significantly in both wild type and the hypersensitive nl strain. at 20°c growth was 10– times slower then the wild type control, slightly more pronounced in the rrf- strain. a large proportion of sterile f animals was also observed. the minority of animals that were fertile produced very few eggs but these eggs were as viable as wild type. according to the previous studies down-regulation of the same gene has produced the following phenotypes: gro, emb, stp, lva. our results for down-regulation of b a are consistent with the data obtained by kamath et al.. rnai resulted in an egl phenotype that was more pronounced in the rrf- background. the involvement of the gene in oviposition was inferred from the mutant phenotype. the gene has orthologous sequences in both the human and mouse genomes that code for a small ctd phosphatase and nuclear lim interactor-interacting factor respectively. rnai on c05c has been reported by simmer et al. as emb, lva and lvl. however, our results are consistent with another study published by kamath et al. where no specific phenotype was observed. the mutant phenotype of c05c points to its involvement in embryonic and larval development with a molecular function associated with protein binding. although a greater proportion of genes show specific rnai phenotypes when using rrf- strain, our results point to an increase in the severity of the phenotypes following down-regulation in the rrf- background, rather than additional phenotypes. subcellular localization in human cell lines for seven of the human orthologs presented in this study, we cloned full-length open reading frames, representing encoded cdnas, into vectors containing v epitope sequences in order to generate recombinant fusion proteins for immunofluorescence detection. because of the novelty of these proteins and the lack of available antibodies against such proteins, it was necessary to overexpress in human cell lines and subsequently detect with antibodies against the v fusion tag. subcellular localizations were obtained in two human cell lines: hela and human embryonic kidney. endogenous expression for the proteins studied in the two cell lines was confirmed by rt-pcr. np_ human ortholog for zk, displays three distinct patterns: diffuse nuclear, nucleolar and nuclear foci. we were able to confirm the nucleolar pattern by colocalization with fibrillarin, a protein predominantly found in nucleoli and cajal bodies. we were unable to determine the exact sub-compartment to which the nuclear foci belong. however, the pattern is strikingly similar to that of paraspeckles. the proteins, described as u snornp protein homologs, belong to the imp family of proteins which are small ribonucleoproteins involved in pre-ribosomal rna processing. these results are consistent with those previously obtained in yeast and recently described in human. based on the interaction of these proteins with snorna in 60-80s rnp complexes and their likely involvement in pre-rrna processing, it is possible to speculate that the preferred location of the imp protein is both transient and dynamic throughout the nucleus, which would explain the three patterns we observe. np_ also described as hypothetical protein mgc and predicted ortholog of worm protein c17e displayed a nuclear and perinuclear pattern covering the entire circumference of the nucleus but did not appear to be inside the nucleus. we were able to confirm the presence of this protein at the nuclear membrane by colocalization with lamin, denoting a possible role for this protein as part of the nuclear envelope. c3hc which contains a ring-finger motif, has been classified as the third member of the recently defined membrane-associated ring-ch protein family. the same authors reported that proteins belonging to this family, including march-iii, characteristically contain two predicted c-terminal transmembrane domains, indicating a possible association with membrane-bound organelles. furthermore, bartee et al. concluded that localization of human march iii by fusion protein overexpression revealed a punctate pattern partially overlapping with cytoplasmic vesicles, specifically early endosomes. however, we were unable to confirm this finding. it is important to note that proteins are, in many cases, dynamic in location meaning that there are potentially multiple locations. thus, our co-staining data are the most compelling we have seen. aak25825/np_ also described as btb/poz domain containing, exhibited a cytoplasmic pattern resembling elongated "worm-like" bodies for which we were unable to determine a known structure. members of this domain family have been shown to interact with co-repressor complexes involved in transcriptional repression. previously, btbd has been shown to interact specifically with topoisomerase i. in addition, a recent study further characterized btbd as colocalizing with trim family members. interestingly, trim proteins have been shown to exhibit ubiquitin ligase activity. np_ has no functional annotation and has only been described as an open reading frame located on human chromosome this protein exhibits weak expression when "overexpressed" under control of cmv promoter. immunofluourescence shows this protein to have both a cytoplasmic and a nuclear distribution. furthermore, the cytoplasmic expression appears punctate and partially colocalizes with calreticulin, a marker for endoplasmic reticulum. aap /jc is a member of a family of small c-terminal domain phosphatase and contains an nif domain. other members of this family have been shown to interact with rna polymerase ii and show nuclear localization. surprisingly, scp was detected at the plasma membrane by colocalization with annexin ii, a known component of the plasma membrane. in addition to this pattern, we noted fibrous structures that partially colocalized with beta-actin. hypothetical protein np_ expression was detected by western blot and immunofluorescence microscopy. microscopy detection displayed a cytoplasmic/nuclear rim staining pattern. however, western blot analysis revealed a molecular weight of approximately kda which is inconsistent with the predicted size of kda, indicating a possible premature stop in translation or post-translational cleavage. in addition, we were unable to detect ectopic expression for np_ any attempts to detect this protein via western blot or microscopy were unsuccessful despite sequence validation of the corresponding expression plasmid.  specific expression patterns have been identified for the f41d, zk, c17e and c09d gfp-fusion strains. visible rnai phenotypes for t24d, f23c, zk and b a were observed. rnai on t24d a produced an extensive phenotype that was not revealed in the large-scale study by fraser et al.. thus, our study shows the value of analyzes focusing on a small number of candidate genes where a variety rather then a pre-defined set of phenotypes are observed. in addition, immunofluorescence microscopy of human cell lines over-expressing np_ np_ aak25825/np_ np_ aap /jc and np_ has detected subcellular localization of the corresponding proteins. the most complete data set comprising both a visible rnai phenotype or/and a distinct gfp expression in c. elegans and a subcellular protein over-expression profile using human cell lines has been obtained for zk, f41d and c17e. in the case of genes f41d and c17e, a mainly neuronal gfp expression was detected. therefore, the absence of an observable rnai phenotype may be explained by the known refractory nature of rnai in this tissue. for the gene zk, which showed a more generalized effect on the development, corresponding widespread expression pattern was observed in the worm and in human cell lines. given that the protein prediction indicates this gene as a snornp candidate, its involvement in a variety of cellular processes thus can be expected. to the contrary, restricted subcellular expression patterns in human cells observed in a few cases, for example the aak and np_115683c11d genes, correlate with the absence of phenotypes other than the wild type. this fact suggests that these genes may be of somewhat lesser importance. in conclusion, this comparative c. elegans-human cell lines study based on orthology assignments explores gene functionality by combining two key aspects of functional genomics: a whole organism approach and protein overexpression data using cells lines. our results extend currently available information on the selected genes providing a step more towards identifying their exact function. 
because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as rnai screens, can be rapidly tested. in order to explore the function of a set of c. elegans genes of unknown function, as well as their potential functional roles in the human genome, we performed a phylogenetic analysis to select the most probable worm orthologs. a total of c. elegans genes were subjected to down- regulation via rnai and characterization of expression profiles using gfp strains. in addition, subcellular protein over-expression profiles of the human orthologs for seven out of the thirteen genes using human cells were also analyzed. many aspects of the nematode caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. by combining a whole-organism approach using c. elegans with complementary experimental work done on human cell lines, this analysis extends currently available information on the selected set of genes. previously unknown distinct expression patterns were observed for four of the analyzed genes, as well as four visible rnai phenotypes.
 many aspects of the nematode caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as rnai screens, can be rapidly tested.  in order to explore the function of a set of c. elegans genes of unknown function, as well as their potential functional roles in the human genome, we performed a phylogenetic analysis to select the most probable worm orthologs. a total of c. elegans genes were subjected to down- regulation via rnai and characterization of expression profiles using gfp strains. previously unknown distinct expression patterns were observed for four of the analyzed genes, as well as four visible rnai phenotypes. in addition, subcellular protein over-expression profiles of the human orthologs for seven out of the thirteen genes using human cells were also analyzed.  by combining a whole-organism approach using c. elegans with complementary experimental work done on human cell lines, this analysis extends currently available information on the selected set of genes. 