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The adoption of biomedical signals such as photoplethysmogram (PPG) and electrocardiogram (ECG) for health parameter estimation on wearable devices is growing in tandem with the increase of attention in mobile healthcare. In our work, we use PPG signals extracted from PPG sensors which are used for biometric identification. A challenge for biometric identification using PPG signal is the variation in domain (placement of sensors, wavelengths, device variation, etc.). In this work, we propose the use of both unsupervised and semi-supervised adversarial learning techniques for cross-domain adaptation. As such algorithm will be deployed on wearable devices, we propose a compact model meeting tight memory footprint limitation. All experiments will be simulated using a public dataset (TROIKA) and our in-house dataset. By introducing a cross-domain adaptation approach across sensors, we observe an accuracy gain of 4.15% on our in-house dataset. The proposed semi-supervised learning technique gives an additional accuracy boost of 2.02%.

The ubiquitin-specific protease USP7 stabilizes both Mdm2 and p53 by removing ubiquitins, hence playing an important enzymatic role in the p53-Mdm2 pathway. However, it is poorly understood how USP7 executes its dual-stabilization effect on Mdm2 and p53 in cellular context. Here, we report that STIP is a novel macromolecular scaffold that links USP7 to the p53-Mdm2 pathway. STIP and a fraction of USP7 interact and constitutively colocalize in nucleoplasma. Overexpression of STIP stabilizes Mdm2 and p53, whereas downregulation of STIP decreases Mdm2 and p53 levels. The effect of STIP on Mdm2 and p53 depends on USP7 function as a deubiquitinating enzyme. Furthermore, we demonstrate that STIP mediates the assembly of two separate ternary protein complexes in vivo as STIP-USP7-Mdm2 and STIP-USP7-p53, which facilitates USP7-mediated stabilization of Mdm2 and p53. Collectively, these results pinpoint a new molecular function of STIP and reveal a novel mechanism whereby USP7 executes its dual-stabilization effect on Mdm2 and p53 via STIP scaffolding.

Global climate changesfrequently cause severe floodingwhich damage peoples propertiesand livesin Taiwan. As a result, the Taiwan government initiated a long-term plan to prevent flooding by regulating 130 rivers in flood-prone areas. However, the budget is limited and cannot meet the total cost of the hydraulic engineering of the 130 rivers. How to objectively and efficiently prioritize rivers that need to be regulated remains a major challenge for the government. This research collected the information of regulating 130 rivers in terms of their costs and benefits; then it utilized a hybrid method that integratesboththedynamic programming and the Paretofront analysis (designated as DPPF here) to develop a budget allocation model.The results show that this model can help the government to save up to NT$ 2.153 billionannually.

Coordinated regulation of fat storage and utilization is essential for energy homeostasis, and its disruption is associated with metabolic syndrome and atherosclerosis in humans. Across species, Krüppel-like transcription factors (KLFs) have been identified as key components of adipogenesis. In humans, KLF14 acts as a master transregulator of adipose gene expression in type 2 diabetes and cis-acting expression quantitative trait locus associated with high-density lipoprotein cholesterol. Herein we report that, in Caenorhabditis elegans, mutants in klf-3 accumulate large fat droplets rich in neutral lipids in the intestine; this lipid accumulation is associated with an increase in triglyceride levels. The klf-3 mutants show normal pharyngeal pumping; however, they are sterile or semisterile. We explored important genetic interactions of klf-3 with the genes encoding enzymes involved in fatty acid (FA) β-oxidation in mitochondria or peroxisomes and FA synthesis in the cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase (fat-7). We show that mutations or RNA interference in these genes increases fat deposits in the intestine of acs-1, acs-2, F08A8.1, and F08A8 animals. We further show that acs-1 and F08A8.1 influence larval development and fertility, respectively. Thus, KLF3 may regulate FA utilization in the intestine and reproductive tissue. We demonstrate that depletion of F08A8.1 activity, but not of acs-1, acs-2, F08A8.2, or fat-7 activity, enhances the fat phenotype of the klf-3 mutant. Taken together, these results suggest that klf-3 regulates lipid metabolism, along with acs-1, acs-2, F08A8.1, and F08A8.2, by promoting FA β-oxidation and, in parallel, may contribute to normal reproductive behavior and fecundity in C. elegans.

Background Over 40 years ago, an unusual Rh phenotype denoted DIVa(C)- was identified in a case of fatal haemolytic disease of the newborn in the third child of Madame Nou. Her RBCs expressed a partial D, weak C and four low-prevalence Rh antigens: Goa (RH30), Rh33 (RH33), Riv (RH45) and FPTT (RH50). The purpose of this study was to determine the molecular basis associated with this rare DIVa(C)- complex. Material and Methods Blood samples were from three donors previously identified as carrying the DIVa(C)- haplotype. Molecular analyses were performed by standard methods. Results The three donors were heterozygous for RHD and RHD*DIVa.2, and all carried a compound hybrid allele at the RHCE locus. This hybrid RHCE allele contained exons 2 and 3 from RHD*DIVa.2 and exon 5 from RHD [RHCE*CE-DIVa.2(2-3)-CE-D(5)-CE] and is in cis to RHD*DIVa.2. The RHCE allele on the in trans chromosome differs between the donors and is RHCE*cE in donor 1, RHCE*ce (254C, 733G) in donor 2 and RHCE*ce in donor 3. Conclusions The RHD*DIVa.2 encodes the Goa antigen, whereas the compound hybrid allele most likely encodes Rh33, Riv and FPTT. The weakly expressed C antigen on RBCs with the DIVa(C)- phenotype could be encoded by exons 2 and 3 from RHD*DIVa.2 in the compound hybrid. This is the first report of RHD*DIVa.2 being involved in a hybrid gene at the RHCE locus. As only one example of anti-Riv has been described, our molecular analysis and findings provide a tool by which to predict Riv expression.

The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor's involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.

In vertebrates, adipose tissue stores energy in the form of fat. Fat storage is tightly controlled by and dynamically balanced with energy expenditure under physiological settings; the perturbation of fat in either excess (obese) or deficit (lipodystrophy) has devastating pathologic consequences in the fueling of homeostasis and organismal fitness. The process by which fat storage is coordinated through positive and negative feedback signals is still poorly understood. To address potential mechanisms underlying fat storage we study a Caenorhabditis elegans Krüppel-like transcription factor, Ce-klf-3 and demonstrate that klf-3 is a hitherto unrecognized key regulator of fat metabolism in C. elegans. The Ce-klf-3 is highly expressed during larval development and predominantly present in intestine: the site of fat digestion, absorption, storage, and utilization. We found a strong positive correlation between klf-3 expression and fat deposition in a worm's intestine. Significantly, a klf-3 (ok1975) loss-of-function mutation, characterized by the deletion of a 1658-bp sequence spanning the 3' end of exon 2 through to the 5' end of exon 3 of klf-3, enhanced fat deposition in the intestine and caused severe defects in worm reproduction. Although klf-3 mutants seemed very similar to wild type worms in appearance and life span, 70% of mutants became semi-sterile, each producing 40-50 viable progenies, and the remaining 30% were rendered completely sterile toward adulthood. Notably, both mutant types displayed extensive deposition of fat in the intestine. Our study also demonstrates that klf-3 is critical for maintaining normal fatty acid composition by regulating genes involved in a fatty acid desaturation pathway. Strikingly, klf-3 mutant animals with impaired fatty acid beta-oxidation pathway genes resulted in fat accumulation in the mutant worm. We present the first clear in vivo evidence supporting essential regulatory roles of KLF-3 in fat storage in C. elegans and shed light on the human equivalent in disease-gene association.

This study examined the effects of NH3-plasma treatment on the gate insulator (SiO2) surface before pentacene deposition. The NH3-plasma treatment can improve the interface property between SiO2/pentacene, providing a suitable surface for pentacene growth. Moreover, the NH3-plasma treatment can also help terminate dangling bonds at the SiO2 surface and thus reduce the interface trap-state density. The proposed method provides a simple and effective method for treating the interface between SiO2/pentacene, reducing interface traps and simultaneously improving pentacene crystallization.

Ser/Thr- and Tyr-Protein kinases constitute a key switch underlying the dynamic nature and graded regulation of signal transduction and pathway activities in cellular organization. Here we describe the identification and characterization of Dusty, a single-copy gene that arose in metazoan evolution and encodes a putative dual Ser/Thr and Tyr protein kinase with unique structural features. Dusty is widely expressed in vertebrates, broadly distributed in the central nervous system, and deregulated in certain human cancers. Confocal imaging of transiently expressed human Dusty-GFP fusion proteins showed a cytoplasmic distribution. Dusty proteins from lower to higher species display an increasing degree of sequence conservation from the N-terminal non-catalytic domain to C-terminal catalytic domain. The non-catalytic region has eight conserved cysteine residues, multiple potential kinase-docking motifs and phosphorylation sites, whereas the catalytic domain is divergent and about equally distant of Ser/Thr and Tyr protein kinases. Homology analyses identified the essential catalytic residues, suggesting that Dusty homologues all possess the enzymatic activity of a protein kinase. Taken together, Dusty is a unique evolutionarily selected group of divergent protein kinases that may play important functional roles in the brain and other tissues of vertebrates.

BACKGROUND: The rare phenotypes Dc- and D-- lack the expression of E/e and CcEe antigens, respectively; their cotransmission in a single family has not been reported. STUDY DESIGN AND METHODS: Six members of a Chinese family with two exhibiting the Dc- phenotype were studied using standard serologic methods. Rh genotypes were analyzed by Southern blot, and RH loci, by exon PCR. Rh transcripts were characterized by gene-specific RT-PCR and sequencing. RESULTS: Although Rh typing detected two members as Dc- homozygotes, RFLP analysis and exon PCR showed them to be Dc- heterozygotes with a partial deletion of RHCE. cDNA sequencing showed the expression in the family of normal RHD and RHCe as well as hybrid transcripts, RHD(1-9)/RHCE(10) and RHCE(1-3)/RHD(4-10). Thus, the Dc- members had the genotype of Dc-/ D-- and expressed both hybrid genes that were inherited from their parents, respectively. DISCUSSION: This is the first demonstration in a family that the Dc- and D-- complexes neither are linked with a normal RHD or RHCE gene. The segregation of these two different hybrid genes with single break points sug-gests their independent genetic origin and provides molecular insights into the dynamic nature of genomic rearrangements leading to RH locus contraction.

BACKGROUND: The S-s-U- phenotype in African Americans is due to a GYPB deletion, however the molecular basis for the S-s-U+var phenotype is poorly understood. Variable reactivity of S-s-U+var RBCs with monoclonal anti-He or by anti-U has been demonstrated, however the underlying molecular bases for this phenotype remain to be established. STUDY DESIGN AND METHODS: Hemagglutination was performed on 104 S-s- blood samples using monoclonal anti-He and anti-U. GYPB was sequenced from selected samples. Allele and exon-specific PCR analysis was used to identify wild-type and mutant alleles. RESULTS: The RBCs of 49-percent S-s- samples were identified as S-s-U+var by hemagglutination. Sequencing analysis of 41 samples revealed 1) a point mutation at +5 (g > t) of intron 5 that resulted in skipping of exon 5 in 34 samples; 2) two mutations (208G > T and 230C > T) caused partial skipping of exon 5 in four samples due to activation of a cryptic 3' splice site that resulted from a C > G transversion at nt251 present in all GYPB*S alleles and most GYPB*s alleles tested. Three samples were heterozygous for the mutated alleles. DISCUSSION: The S-s-U+var phenotype arises from changes in or around GYPB exon 5. The weak expression of U and in most examples, He, may be due to low levels of normal transcription of the variant gene or to posttranscriptional down regulation. (Less)

The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH 3 , we recently speculated that Rh proteins are biological gas channels for CO 2 . Like NH 3 , CO 2 differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii . Expression of RH1 was high for cells grown in air supplemented with 3% CO 2 or shifted from air to high CO 2 (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO 2 ) or shifted from high CO 2 to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO 2 .

Ammonium transporters play a key functional role in nitrogen uptake and assimilation in microorganisms and plants; however, little is known about their structural counterpart in mammals. Here, we report the molecular cloning and biochemical characterization of Rh type B glycoproteins, human RhBG and mouse Rhbg, two new members of the Rh family with distinct tissue specificities. The RhBG orthologues possess a conserved 12-transmembrane topology and most resemble bacterial and archaeal ammonium transporters. Human RHBG resides at chromosome 1q21.3, which harbors candidate genes for medullary cystic kidney disease, whereas mouse Rhbg is syntenic on chromosome 3. Northern blot and in situ hybridization revealed that RHBG and Rhbg are predominantly expressed in liver, kidney, and skin, the specialized organs involving ammonia genesis, excretion, or secretion. Confocal microscopy showed that RhBG is located in the plasma membrane and in some intracellular granules. Western blots of membrane proteins from stable HEK293 cells and from mouse kidney and liver confirmed this distribution. N-Glycanase digestion showed that RhBG/Rhbg has a carbohydrate moiety probably attached at the NHS motif on exoloop 1. Phylogenetic clustering, tissue-specific expression, and plasma membrane location suggest that RhBG homologous proteins are the long sought major ammonium transporters in mammalians.

The past decade has seen extensive studies of the erythrocyte Rh30 polypeptides and Rh-associated glycoprotein, which specify the clinically important Rh blood group system. Here we consider recent advances on these and other Rh homologues in the context of gene organization, molecular evolution, tissue-specific expression, protein structure, and potential biological functions. The Rh family is now known to contain a large number of homologues that form a unique branch in the eucarya life domain. The ancient origin and broad distribution imply central roles for the various Rh proteins in maintaining normal cellular and organismal homeostatic conditions. Rh homologues occur in the form of multiple chromosomal loci in mice and humans, but as single-copy genes in unicellular organisms (e.g., green alga and slime mold). While primitive Rh genes vary largely in exon/intron design, the mammalian Rh homologues bear a similar genomic organization. Sequence comparisons have revealed the signatures and a consensus 12-transmembrane fold characteristic of the Rh family. Phylogenetic analysis has placed all Rh homologues as a related cluster that intercepts ammonium transporter (Amt) clusters, indicating an intimate evolutionary and structural relationship between the Rh and Amt families. The biochemical identification and epithelial expression of RhBG and RhCG orthologues in mammalian kidney, liver, skin, testis, and brain suggest that they serve as transporters likely participating in ammonia homeostasis. Further inquires into the structure, function, biosynthesis, and interaction of Rh proteins will shed new light on ammonia homeostasis in a wide range of human physiological and pathological states.

BACKGROUND: In a Moroccan family, the partial D phenotype DBT is defined by an RHD-CE-D gene in which exons 5 to 7 of RHD were replaced by those of RHCE. In this study, the molecular basis and inheritance of DBT in a Japanese family are described. STUDY DESIGN AND METHODS: A Japanese proposita exhibiting the DBT phenotype was analyzed by serologic methods and molecular techniques. The RH transcripts of the proposita were sequenced and compared with those of normal donors. The inheritance and structure of the RH genes in the family were determined by Southern blot analysis and exon-specific polymerase chain reaction. RESULTS: The proposita typed weak D and C+c+E+e+Rh:32. Family data indicated a cotransmission of Rh32 with DBT and a linkage of C and e with DBT. Southern blot testing of the proposita's genomic DNA indicated a partial and a total absence of RHD on the respective homologous chromosomes. Sequencing of her cDNA showed expression of Ce, cE, and RHD-CE-D transcripts but not D. The RHD-CE-D hybrid was characterized by a conversion of five RHD exons into RHCE exons (exons 5-9). The 5′ and 3′ breakpoints in the fusion gene were localized to the intron 4/exon 5 region and intron 9. CONCLUSION: The new RHD-CE-D gene defines a separate genetic origin of DBT in the Japanese family. The proximal sequence encoded by RHD exon 4 and RHCE exon 5, together with the distal RHCE sequence, may involve the cotransmission of Rh32 and DBT, which behave as codominant and recessive characters, respectively.

Rh(null) is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rh(null) cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rh(null)(HT), RHAG harbored in exon 6 two G-->A transitions, GTT-->ATT and GGA-->AGA, which cause Val(270)-->Ile and Gly(280)-->Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rh(null)(WO), RHAG contained in exon 9 a single G-->T transversion, GGT-->GTT, which caused a Gly(380)-->Val missense change in TM12 segment. The G-->T transversion, which is located at the +1 position of exon 9, had also affected pre-mRNA splicing and caused partial exon skipping. Although both Rh(null) cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein.

To seek an alternative model for studies of theRh protein complex, we isolated by homology cloning andcharacterized the mouse Rhced and Rhaggenes, which are homologous to the human RH andRHAG genes,respectively. Rhced encodes a glycoprotein of418 amino acids which occurs as a composite of human RhDand RhCE with 60% identity and 74% similarity. Rhagencodes a glycoprotein of 438 amino acids thatshares 79% identity and 87% similarity to humanRh50. However, Rhag has an elongated C terminus and fourN-glycosylation sites clustered on exoloop 1. Hydropathyplots suggest that Rhl1 and Rhag each spanthe lipid bilayer 12 times, with N and Ctermini facing the cytoplasm. Rhced and Rhag are bothspecified by 10 exons and bear a similar exon/intronstructure, but their major transcription start sites aremapped at –17A and –27A. Northernanalysis revealed coexpression of Rhced and Rhag from11-day embryos throughout adult life in erythroidtissues. Southern blotting and linkage analysis showedthat Rhced and Rhag are single-copygenes localized to chromosomes 4 and 17, respectively;they are paralogous to one another but orthologous tohuman RH and RHAG. The resultstogether predate the occurrence and signifya conserved function of the erythroid-specificRh membrane structures.

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at theRH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G→A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy intrans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical fromCaenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.© 1998 by The American Society of Hematology.

Rhnull disease, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic hemolytic anemia. We studied here a German family transmitting a putative amorph Rhnull disease gene and identified a rare mutation causing the loss-of-function phenotype. We analyzed the genomic and transcript structure of RH30, RH50, andCD47, the three loci thought to be most critical for expression of the Rh complex in the red blood cell membrane. We showed that in this family the Rh50 and CD47 transcripts were normal in primary sequence. However, the RH30 locus contained an unusual double mutation in exon 7 of the RhCe gene, in addition to a deletion of the RhD gene. The mutation targeted two adjacent codons in multiple arrangements probably via the mechanism of microgene conversion. One scheme entails a noncontiguous deletion of two nucleotides, [ATT(Ile322)→AT] and [CAC(His323)→CC], whereas the other involves a T→C transition [ATT(Ile322)→ATC] and a dinucleotide deletion [CAC(His323)→C]. They caused the same shift in open reading frame predicted to encode a shortened protein with 398 amino acids. The loss of two transmembrane domains and gain of a new C-terminal sequence are likely to alter the protein conformation and impair the Rh complex assembly. Our findings establish the molecular identity of an amorph Rhnull disease gene, showing that Rh30 and Rh50 are both essential for the functioning of the Rh structures as a multisubunit complex in the plasma membrane.

The Rh (Rhesus) protein family comprises Rh50 glycoprotein and Rh30 polypeptides, which form a complex essential for Rh antigen expression and erythrocyte membrane integrity. This article describes the structural organization of Rh50 gene and identification of its associated splicing defect causing Rhnulldisease. The Rh50 gene, which maps at chromosome 6p11–21.1, has an exon/intron structure nearly identical to Rh30 genes, which map at 1p34–36. Of the 10 exons assigned, conservation of size and sequence is confined mainly to the region from exons 2 to 9, suggesting thatRH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event. The available information on the structure of RH50facilitated search for candidate mutations underlying the Rh deficiency syndrome, an autosomal recessive disorder characterized by mild to moderate chronic hemolytic anemia and spherostomatocytosis. In one patient with the Rhnull disease of regulator type, a shortened Rh50 transcript lacking the sequence of exon 7 was detected, while no abnormality was found in transcripts encoding Rh30 polypeptides and Rh-related CD47 glycoprotein. Amplification and sequencing of the genomic region spanning exon 7 revealed a G → A transition in the invariant GT motif of the donor splice site in both Rh50 alleles. This splicing mutation caused not only a total skipping of exon 7 but also a frameshift and premature chain termination. Thus, the deduced translation product contained 351 instead of 409 amino acids, with an entirely different C-terminal sequence following Thr315. These results identify the donor splicing defect, for the first time, as a loss-of-function mutation at theRH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions.

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at −6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

As a partial D antigen of the Rh blood group system, the D category IIIa phenotype occurs mainly in Blacks, but its molecular basis has not been defined. Here we describe studies of the D category D(IIIa) and VS+ red blood cells (RBC) from two unrelated probands by Southern blot, cDNA PCR, and nucleotide sequencing. Rh haplotyping by Sph I restriction fragment length polymorphisms indicated that the two probands carried Dce/dCe and Dce/DcE genotypes, respectively. Sequence analysis of Rh cDNAs showed that their erythroid cells expressed both D and CE transcripts. Nevertheless, the D transcripts were found to contain four nucleotide changes scattered in three exons: nt455 A-to-C (exon 3), nt602 C-to-G (exon 4), nt 654 C-to-G (exon 5), and nt667 T-to-G (exon 5). These variations resulted in the following amino acid substitutions characteristic of RhCE polypeptides: 152 Asn-to-Thr, 201 Thr-to-Arg, 218 Ile-to-Met, and 223 Phe-to-Val. The 152Thr and 223Val residues were predicted to reside in proximity to the third and fourth extracellular loops, respectively. Together, these results establish a correlation of the four amino acid changes in the RhD protein with the expression of D(IIIa) as a partial D antigen on the RBC membrane. Since the varied nucleotides identified in D(IIIa) all pre-exist in CE, they are likely to have originated from CE by templated micro-conversion event(s). The identification of a specific nt736 C-to-G transversion in CE in the two probands suggests that 245Val may involve the expression of VS antigen.

Homologues of MN blood group antigens, encoded by members of the glycophorin A (GPA) gene family, are expressed in man, anthropoid apes, and some species of Old World monkeys. Previous studies had shown that a three-gene framework, most closely related to that in man, is present in the chimpanzee. Here we report the genomic structure, transcript map, and protein expression of the GYPA locus in gorillas. Compared to the corresponding human and chimpanzee homologues, gorilla GPA, GPB, and GPB/E genes each showed a high degree of sequence identity, with the same exon-intron organization. However, the expression of exons III, IV, or V encoding the extracellular or membrane domains of homologous glycophorins varied among the three species. Gorilla GPA and GPB/E genes were unique in that the former occurred in two allelic forms with or without the expression of exon III, whereas the latter contained one (ψ exon III) instead of two silenced exons (ψ exons III and IV). Differences from human but not chimpanzee GPA also included the presence of a hybrid M/N epitope and the absence of the sequon for N-glycosylation. Owing to the retention of a functional exon III, gorilla GPB was more similar to chimpanzee GPB than human GPB. A transspecies allele was identified in the gorilla that gave rise to the Henshaw (He)-like antigen similar to that found in man. These results provide further insight into the model for evolution of the GPA gene family, indicating that the mechanisms underlying inter- and intraspecific polymorphism of glycophorins could predate the divergence of gorillas as the consequence of gene duplication and diversification.

Because cardiovascular disease remains a serious problem in modern human society, the aim of this study was to establish the rat model animal and to compare the heart dysfunction and fibrosis with SD and LE rats when treated with myocardial ischemia and reperfusion operation. A 20-minute thoracotomy was performed on the rat at the left anterior descending coronary artery occlusion; then the perfusion was carried out. The left ventricular diastolic diameter and left ventricular systolic diameter (LVEDd and LVEDs) were both reduced in LE and SD rats after surgery. Compared with the sham group, the performance of the left ventricular fractional shortening (LVFS) significantly decreased, indicating systolic dysfunction was affected after surgery, and SD was significantly higher than LE at LVFS decreasing rate. The significant areas of collagen fibers were detected by Masson's tri-chrome staining after surgery. These results suggest that SD rat is more suitable than LE rat for successful establishment of the model of myocardial ischemia and reperfusion. Also, the rat model can provide good experimental materials for regenerative medicine and drug testing to enhance research results in the future. Key words : Myocardial infarction, myocardial ischemia and reperfusion, animal model.

This report describes a comparative study on the dCCee and DCW-red blood cells devoid of RhD and CcEe antigens, respectively. Southern blots showed that the two variants carried opposite deletions in the D and non-D (CcEe) genes. Rh haplotyping and exon polymerase chain reaction (PCR) assay indicated that the deletions did not extend beyond the 5′ region upstream from exon 1 or the 3′ region downstream from exon 10 of the respective genes. This was confirmed by finding intact promoters and 3′ untranslated regions in both D and non-D genes in each variant. Reverse transcriptase-PCR and cDNA sequencing showed the expression of two transcripts in each cell type. In dCCee cells, one transcript was the regular Ce form and the other occurred as a D-Ce-D hybrid whose Ce sequence spanned exons 2 through 9. In DCW-cells, the two transcripts were derived from reversely arranged hybrid genes, ie, the CW-D gene was formed by fusion of CW exon 1 with D exons 2 through 10, whereas the reverse product was formed by fusion of D exons 1 through 9 with non-D exon 10. These results indicated that DNA deletion and recombination had occurred in either cis or trans configuration and involved both RH loci in the dCCee or DCW-genome. Identification of such compound alterations correlates the genotypes with phenotypes and explains the lost Rh antigenic expression. A reinvestigation of gene organization also led to the reassignment of several 5′ and 3′ splice sites. Together, this study not only shows the complexity of Rh phenotypic diversity, but also points to the importance of concurrent analysis of genomic structure and transcript expression in deciphering the underlying genetic mechanisms.

The Wright (Wra/Wrb) blood group polymorphism is defined by an allelic change (Lys658Glu) in the band 3 protein; nevertheless, the Wrb antigen apparently requires glycophorin A (GPA) for surface presentation. To gain insight into the structural basis for this protein-protein interaction and delineate its relationship with Wrb antigen expression, we investigated GPA and band 3 sequence polymorphisms occurring in rare humans and nonhuman primates. The lack of GPA or amino acid residues 59 through 71 of GPA results in the absence of Wrb from human red blood cells (RBCs) exhibiting the MkMk, En(a-), or MiV phenotype. However, the SAT homozygous cells carried a Glu658 form of band 3 and a hybrid glycophorin with the entire GPA extramembrane domain from residues 1 through 71, yet expressed no Wrb antigen. This finding suggests that formation of the Wrb antigenic structure is dependent on protein folding and that the transmembrane junction of GPA is important in maintaining the required conformation. Comparative analyses of GPA and band 3 homologues led to the identification in the interacting regions of conserved and dispensable amino acid residues that correlated with the Wrb positive or negative status on nonhuman primates. In particular, the chimpanzee RBCs cells expressed Wrb and the Glu658 form of band 3, which is identical to humans, but their GPA contained the Gly rather than Arg residue at position 61. Taken together, the results suggest that (1) Arg61 of GPA and the proposed Arg61-Glu658 charge pair are not crucial for Wrb antigen exhibition and (2) the role of GPA for interaction with band 3, including Glu658, probably involves a number of amino acid residues located in the alpha-helical region and transmembrane junction.

In humans, the allelic diversity of MNSs glycophorins (GP) occurs mainly through the recombinational modulation of silent exons (pseudoexons) in duplicated genes. To address the origin of such a mechanism, structures of GPA, GPB, and GPE were determined in chimpanzee, the only higher primate known to have achieved a three-gene framework as in humans. Pairwise comparison of the chimpanzee and human genes revealed a high degree of sequence identity and similar exon-intron organization. However, the chimpanzee GPA gene lacks a completely formed M- or N-defining sequence as well as a consensus sequence for the Asn-linked glycosylation. In the case of the GPB gene, exon III is expressed in the chimpanzee but silenced, as a pseudoexon, in the human. Therefore, the protein product in the chimpanzee bears a larger extracellular domain than in the human. For the GPE genes, exon III and exon IV have been inactivated by identical donor splice-site mutations in the two species. Nevertheless, the chimpanzee GPE-like mRNA appeared to be transcribed from a GPB/E composite gene containing no 24-bp insertion sequence in exon V for the transmembrane domain. These results suggest a divergent processing of exonic units from chimpanzee to human in which the inactivation of GPB exon III preserved a limited sequence repertoire for diversification of human glycophorins.

The D--phenotype of the human erythrocyte is a genetic variant of the Rh blood group system associated with the expression of D but not C, c, E, and e (designated non-D) antigens. In this report, we characterize the structure and expression of Rh polypeptide genes in two D-- homozygotes of Italian origin. Southern blot analysis detected a gross deletion in their genomic DNA that correlated with the alteration of CcEe rather than the D polypeptide gene. With detailed exon mapping, the deletion was found to be partial and internal, encompassing exons 2 through 8 of the non-D gene. Analysis of Rh cDNAs showed that no functional mRNA was produced from the truncated non-D gene, whereas the D gene gave rise to one major and two minor mature transcripts. The full-length RhD cDNA sequence contained four nucleotide changes resulting in four amino acid substitutions on the polypeptide backbone. The shortened RhD cDNAs occurred as alternatively spliced isoforms lacking sequences corresponding to exons 7 and/or 8. The identification of a partial and internal deletion in the non-D gene shows that the molecular basis for the D--phenotype is heterogenous and that its alterations have occurred on different genetic backgrounds.

Previous studies of two unrelated Japanese families showed that two isoforms of glycophorin were associated with the expression of SAT antigen on the erythrocyte membrane. Here we report the molecular basis for one form of this MNSs-related surface marker that displayed an altered immunoblotting pattern. Evidence is presented that glycophorin SAT (GPSAT) is encoded by a hybrid gene resulting from unequal homologous recombination between GPA and GPB genes. Analysis of SAT genomic DNAs by Southern blots showed gross alterations in the glycophorin gene cluster. Those restriction fragments characteristic of the GPA 3′ and GPB 5′ ends were absent from the SAT homozygote and showed reduced intensity in SAT heterozygotes. Reticulocyte RNA polymerase chain reaction showed the presence in the SAT homozygote of GPSAT and GPE transcripts but no GPA and GPB transcripts. Direct sequencing of the amplified SAT cDNA showed that its sequence from exon I to exon IV was identical with the N allele of GPA, whereas its 3′ portion, including exon V and exon VI, was derived from the GPB gene. The GPSAT protein in its mature form should contain 104 amino acid residues and bear a novel sequence, Ser-Glu-Pro-Ala-Pro-Val, encoded by the junction of GPA exon IV and GPB exon V. This sequence interfaces the extracellular and transmembrane domains and could be the epitope site of the SAT antigen. The formation of such a hybrid junction not only explains why SAT homozygous erythrocytes lack S, s, and U antigens but also shows a reciprocal arrangement with respect to the B-A hybrid GPDantu gene.

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteinsexhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSsblood groups; thus individuals bearing variant glycophorins can be readily identified by serologicaltyping. Examination of the wide array of variants of these antigens showed that they include manyforms, possibly made evident by lack of constraints due to the apparent dispensability of the parentmolecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversionscoupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature isthe shuffling of sequences within two specific exons (one of which is silent), homologous in the twoparent genes. This has resulted in expression of a mosaic of sequences within this region, leading topolymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique amonghuman gene families. It could have occurred by chance rearrangements among closely linked genes andbeen driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibilitycomplex (MHC). In the glycophorin family the small size of the region within which gene interactionshave occurred and the participation of essentially only two alleles makes this relatively simpler systemmore focused and easier to dissect and describe molecularly. © 1995 Wiley-Liss, Inc.

This paper describes the identification in S-s-U-erythrocytes of a novel glycophorin (GP), He(P2), with structural variations in both its extracellular and transmembrane domains. In the exon II-intron 2 region, a sequence transfer from GPA to GPB, probably via the mechanism of gene conversion, was associated with the induction of multiple untemplated nucleotide replacements. These changes defined the sequence for the He epitope while concomitantly abolishing GPB-associated N antigenicity. Moreover, the GPHe(P2) gene carries two splice site mutations that coordinately affect the processing of exon V coding for the transmembrane segment. The C-->G transversion at the 3' end of exon V created a cryptic acceptor splice site, whereas the G-->T transversion at the +5 position of intron 5 altered the consensus of the donor splice site. Transcript sequencing revealed that neither site was utilized in the splicing of GPHe(P2) pre-mRNA. Rather, complete skipping of exon V and subsequent joining of exon IV to exon VI caused a shift in the open reading frame, which remodeled GPHe(P2) with an elongated new hydrophobic sequence for membrane anchoring. As a result, GPHe(P2) does not display the S and U epitopes although it still contains an intact linear sequence for the two antigens. These findings illustrate how exon and intron sequences concertedly determine the specificity of in vivo splice site selection. In addition, they pinpoint the conformational dependence of the S, s, and U antigens and the importance of the hinge region for their presentation.

We report the identification in human glycophorin A gene of a novel exon mutation that affects splice site selection. DNA mapping showed that the mutation has abolished a unique MspI site marking the exon III-intron 3 splice junction (ACCG/GT). Genomic sequencing confirmed the occurrence of a single G–>A transition at the terminal nucleotide position of exon III. Analysis of the mRNA composition demonstrated a partial inactivation of the altered 5' splice site as well as skipping of exons involving the alternative use of other constitutive splice sites. The full-length transcript with the cognate A change encodes a variant glycophorin with an arginine replacing a glycine at position 59 and defining the ERIK epitope, whereas the exon III-deleted transcript specifies a shorter glycophorin carrying the Sta antigen. Also identified were the misspliced mRNA species with exon I-IV and exon I-V connections generated by selection of 5' splice sites far distant from the mutated site. Although having a correct translation frame, the predicted polypeptides of such exon-skipping products were not assembled on the erythrocyte membrane probably due to the severe truncation of the signal sequence required for targeting and translocation. These observations reveal a new mechanism for antigenic diversity of human glycophorins.

We report here the pre-mRNA splicing of the human glycophorin Mz (HGpMz) gene altered by a defective donor splice site introduced into the third intron via the mechanism of gene conversion. We found that the directional transfer from HGpB(delta) to HGpA(alpha) of a 145-base pair silent homologous segment had resulted in HGpMz as an alpha-beta-alpha hybrid gene with a G-T transversion in the consensus GT motif of the 5'-donor splice site. Transcript analysis showed the presence in the Mz reticulocytes of three major glycophorin cDNA species of which two occurred as the shortened and aberrantly spliced products of HGpMz pre-mRNA lacking the sequences encoded by one and two exons, respectively. The skipping of exon III results in one HGpMz protein with 99 amino acids that bears the M and Sta blood group antigens, whereas the coincident skipping of both exon III and exon IV leads to the expression of a minor HGpMz protein with 86 amino acids. These results indicate that the translocated defective donor splice site not only causes uniform skipping of the upstream exon but also affects the processing of the downstream exon. Complementing our previous studies, the finding that the active and inactive splice sites can be transposed by DNA recombination and alternatively used to construct new glycophorin alleles reveals a novel mechanism for sequence diversification in human genes.

The human glycophorin (HGp) loci that define the red blood cell surface antigens of the MNSs blood group system exhibit considerable allelic variation. Previous studies have identified gene conversion events involving HGpA(alpha) and HGpB(delta) that produced delta-alpha-delta hybrid genes which differ in the location of breakpoints. This report presents the molecular analysis of HGpMilX, the first example of a reverse alpha-delta-alpha hybrid gene that specifies a newly described phenotype of the Miltenberger complex. A novel restriction fragment unique to the HGpMilX gene was detected by Southern blot hybridization. The structure of the genomic region encoding the entire extracellular domain of the MilX protein was determined. Nucleotide sequencing of amplified genomic DNA showed that a silent segment of the HGpB(delta) gene had been transposed to replace the internal part of exon III in the HGpA(alpha) gene, thereby resulting in the formation of the MilX allele with an alpha-delta-alpha configuration. The proximal alpha- delta breakpoint was found to be flanked by a direct repeat of the acceptor splice site, whereas the distal delta-alpha breakpoint was localized to a palindromic region. This DNA rearrangement, with a minimal transfer of 16 templated nucleotides and a single mutation of untemplated adenyl nucleotide, not only created two intraexon hybrid junctions but transactivated the expression of a new stretch of amino acid residues in the MilX protein. Such a segment replacement may have occurred through the directional transfer from one duplex to the other via the mechanism of gene conversion. The occurrence of HGpMilX as another hybrid derived from parts of parent genes underlines the role of the recombinational “hotspot” in the generation of allelic diversity in the glycophorin family.

The glycophorin locus (GYP) on the long arm of chromosome 4 encodes antigens of the MNSs blood group system and displays considerable allelic variation among human populations. The genomic structure and organization of a variant glycophorin allele specifying a novel Miltenberger (Mi)-related phenotype, MiX, were examined. This variant probably arose from a gene conversion event involving a direct repeat of the acceptor splice site. Southern blot analysis indicated that MiX gene derived its 5' and 3' portions from glycophorin B or delta gene but its internal part from glycophorin A or alpha gene. Genomic sequences encompassing the rearranged regions of the MiX gene were amplified by single copy polymerase chain reaction. Direct DNA sequencing showed that during the formation of MiX gene, a short stretch of alpha exon III with a donor splice site has replaced a silent sequence in the delta gene containing a cryptic acceptor splice site. The upstream delta-alpha breakpoint is flanked by the direct repeats of the acceptor splice site, whereas the down-stream alpha-delta breakpoint is located in the adjacent intron. This segmental transfer produced a new composite exon whose expression not only transactivated a portion of silent sequence but also created intraexon and interexon hybrid junctions that characterize the antigenic specificities of MiX glycophorin. The identification of MiX as yet another delta-alpha-delta hybrid different from MiIII and MiVI in gene conversion sites suggests that shuffling of expressed and unexpressed sequences through particular genomic DNA motifs has been an important mechanism for shaping the antigenic diversity of MNSs blood group system during evolution.

This chapter has reviewed the nature of antigens of the MNSs blood group system. The structures of the proteins and the molecular features and organization of glycophorin genes were described, emphasizing their domain arrangement and the extensive sequence homology that indicates that their common and variant alleles belong to a single gene family. Methods currently used to examine these antigens are immunoblotting and DNA typing. The majority of variant genes are hybrids of parent glycophorin genes in a variety of arrangements; they contain no other sequences but those of the parent genes. The structures of the hybrids are summarized in Figure 8. Several hybrids appear to have arisen by unequal homologous recombination but others appear to have occurred through gene conversion. In this system the molecular genetic basis for a single variant phenotype may differ, as documented by gene rearrangements that appear to have occurred, as separate events, at different sites in the same intron; this has resulted in protein structures (hence phenotypes) that are identical. For example, unequal homologous recombination occurring within intron 3 can have given rise to only a limited number of phenotypes, namely alpha M-delta S, alpha N-delta S, alpha M-delta S, alpha N-delta S and delta-alpha. In addition, different sites of an exon may have been involved in gene rearrangements through gene conversion leading to nearly identical protein structures, yet different serological phenotypes. Thus, gene conversion could be more significant for generation of antigenic diversification as the number of possible new alleles is quite large. The participation of the HGpE gene in these rearrangements would make this number even larger. New sites and the expressed pseudoexon have created the epitopes of the variant phenotypes, and sequences specific for several variant antisera have been identified. Thus, the molecular basis for several serological reactions involving this system is now better understood.

MiV, MiV(J.L.), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between alpha and delta glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L.), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing- over site and showed that MiV and MiV(J.L.) genes are arranged in the same 5′ alpha-delta 3′ frame whereas Sta gene is in a reciprocal 5′delta-alpha 3′ configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The alpha-delta breakpoints in MiV and MiV(J.L.) genes were localized to the 3′ end of the HindIII site downstream from exon 3 and to the 5′ end immediately upstream from exon 4, respectively, whereas the delta-alpha breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L.) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the delta glycophorin alleles that must have participated in the recombination.

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.

Human erythrocyte membrane alpha and delta glycophorins (glycophorins A and B) carry the antigens for the M,N,S,s blood group system. Synthetic oligonucleotides spanning coding regions for M,N,S, and s epitopes were used to examine DNAs from 50 individuals selected at random and from individuals known to exhibit S(-)s(-)U- or S(-)s(-)U+ blood group phenotypes. We showed that M,N,S,s, blood group-specific sequences occur as multicopies in the human genome and reside within the alpha and delta glycophorin genes and also within the third glycophorin gene (glycophorin E gene). DNA typing with M- and N-epitope-specific probes showed distinct patterns that allowed correlation of the genotypes with the blood group phenotypes. The correlation using S- and s-specific probes was less definite owing to cross-hybridization. An Mspl restriction fragment length polymorphism (RFLP) residing in the E gene was detected in the black population. This RFLP is also carried by all individuals tested who exhibit the S(-)s(-)U- and S(-)s(-)U+ blood groups phenotypes, thereby serving as a useful marker for the S-s- alleles. The site of cleavage resulting in this RFLP was localized to the second intron of the E gene, and cleavage could occur through differential methylation of its two alleles.

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

Rhesus (Rh) proteins share a conserved 12-transmembrane topology and specify a family of putative CO 2 channels found in diverse species from microbes to human, but their functional essentiality and physiological importance in metazoans is unknown. To address this key issue and analyze Rh-engaged physiologic processes, we sought to explore model organisms with fewer Rh genes yet are tractable to genetic manipulations. In this article, we describe the identification in nematodes of two Rh homologues that are highly conserved and similar to human Rh glycoproteins, and we focus on their characterization in Caenorhabditis elegans . RNA analysis revealed that CeRh1 is abundantly expressed in all developmental stages, with highest levels in adults, whereas CeRh2 shows a differential and much lower expression pattern. In transient expression in human cells, both CeRh1 and CeRh2-GFP fusion proteins were routed to the plasma membrane. Transgenic analysis with GFP or LacZ-fusion reporters showed that CeRh1 is mainly expressed in hypodermal tissue, although it is also in other cell types. Mutagenesis analysis using deletion constructs mapped a minimal promoter region driving CeRh1 gene expression. Although CeRh2 was dispensable, RNA interference with CeRh1 caused a lethal phenotype mainly affecting late stages of C. elegans embryonic development, which could be rescued by the CbRh1 homologue from the worm Caenorhabditis briggsae . Taken together, our data provide direct evidence for the essentiality of the CeRh1 gene in C. elegans , establishing a useful animal model for investigating CO 2 channel function by cross-species complementation.