TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.     

Cloning and expression of CSAL2, a new member of the spalt gene family in chick

In this study we describe cloning and expression of CSAL2, a second member of the spalt gene family in chick. All spalt proteins are characterized by the presence of multiple zinc-finger motifs, which are highly conserved. Mutations in HSAL1, a human spalt gene result in Townes-Brocks syndrome (TBS). We show here that CSAL2 is expressed in many of the tissues affected in TBS, including neural tissue, limb buds, mesonephros and cloaca.     

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos

 The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation. Received: 19 October 1998 / Accepted: 16 February 1999     

CD300 antigen like family member G: A novel Ig receptor like protein exclusively expressed on capillary endothelium

We report the characteristics of CD300LG, a member of the CD300 antigen like family. Its genomic structure is similar in both mouse and human, and at least four isoforms exist in both species. The amino acid sequence of the immunoglobulin (Ig) V like domain of CD300LG showed approximately 35% identity to those of the polymeric Ig receptor (pIgR) and Fcalpha/muR. Interestingly, mouse CD300LG proteins were uniquely expressed on capillary endothelium. Immunoelectron microscopy revealed that mouse CD300LG is localized on both apical and basolateral plasma membranes, as well as on intracellular vesicular structures, in the capillary endothelium. Transcytosis assays using polarized MDCK epithelial cells showed that CD300LG could be transcytosed bidirectionally. Furthermore, CD300LG exogenously expressed on HeLa cells could take up IgA2 and IgM, but not IgG. These results suggest that CD300LG might play an important role in molecular traffic across the capillary endothelium.     

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.     

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX(2)CX(4)HX(4)C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.     

Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis

We have used bioinformatic data mining to identify a novel, endothelial-specific gene encoding a protein with homology to the axon guidance protein roundabout (ROBO1). The new gene has been called magic roundabout (ROBO4; GenBank acc. no. AF361473) and is smaller than other members of the roundabout gene family. Thus, in the extracellular region, magic roundabout has only two of the five immunoglobulin and two of the three fibronectin domains present in other roundabout genes. Expression of magic roundabout in vitro was detected in only endothelial cells and was greater in cells exposed to hypoxia. In situ hybridization and immunohistochemistry validated the bioinformatic prediction that magic roundabout expression would be endothelial specific in vivo. Magic roundabout expression in the adult was restricted exclusively to sites of active angiogenesis, notably tumor vessels. The identification of magic roundabout shows that the roundabout gene family extends beyond neuronal tissue and that roundabout/slit interactions are likely to have a role in angiogenesis.     

Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain

Screening of a rat brain cDNA library with a radiolabeled probe made from an alpha 3 cDNA (Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemanns, S., and Patrick, J. (1986) Nature 319, 368-374) resulted in the isolation of a clone whose sequence encodes a protein, beta 3, which is homologous (40-55% amino acid sequence identity) to previously described neuronal nicotinic acetylcholine receptor subunits. The encoded protein has structural features found in other nicotinic acetylcholine receptor (nAChR) subunits. Two cysteine residues that correspond to cysteins 128 and 142 of the Torpedo nAChR alpha subunit are present in beta 3. Absent from beta 3 are 2 adjacent cysteine residues that correspond to cysteines 192 and 193 of the Torpedo subunit. In situ hybridization histochemistry, performed using probes derived from beta 3 cDNAs, demonstrated that the beta 3 gene is expressed in the brain. Thus, beta 3 is the fifth member of the nAChR gene family that is expressed in the brain. The pattern of beta 3 gene expression partially overlaps with that of the neuronal nAChR subunit genes alpha 3, alpha 4, or beta 2. These results lead us to propose that the beta 3 gene encodes a neuronal nAChR subunit.     

A new member of the insulin receptor family, insulin receptor-related receptor, is expressed preferentially in the kidney.

Shier and Watt isolated human and guinea pig genomic DNA encoding a putative protein, insulin receptor-related receptor (IRR), the primary structure of which is similar to that of other members of the insulin receptor family, the insulin receptor and type-I IGF receptor (J. Biol. Chem. 264, 14605-14608 (1989)). However, the expression of the IRR gene remained unknown. In this paper, we isolated the IRR cDNA from the rat brain and examined the expression of the IRR mRNA in a variety of rat tissues, including the brain, heart, lung, liver, small intestine, kidney, thymus, spleen, muscle, adipose tissue and cartilage by polymerase chain reaction. In contrast to the wide distribution of the insulin receptor and type-I IGF receptor mRNAs, the IRR mRNA is expressed preferentially in the kidney, which indicates that IRR has unique functions as a member of the insulin receptor family.     

A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin

Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.     

Foxn4--a new member of the forkhead gene family is expressed in the retina

We report the cloning and expression of a novel murine forkhead/winged helix family member--Foxn4--that is expressed during neural development in the retina, the ventral hindbrain and spinal cord and dorsal midbrain. Retinal Foxn4 expression is associated with the zone of proliferating progenitor cells. In the mouse mutant ocular retardation (or(J)), Foxn4 expression in the retina is significantly reduced and terminates prematurely.     

A new member of the NY-ESO-1 gene family is ubiquitously expressed in somatic tissues and evolutionarily conserved

Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.     

Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.     

AtREM1, a member of a new family of B3 domain-containing genes, is preferentially expressed in reproductive meristems

We have isolated and characterized AtREM1, the Arabidopsis ortholog of the cauliflower (Brassica oleracea) BoREM1. AtREM1 belongs to a large gene family of more than 20 members in Arabidopsis. The deduced AtREM1 protein contains several repeats of a B3-related domain, and it could represent a new class of regulatory proteins only found in plants. Expression of AtREM1 is developmentally regulated, being first localized in a few central cells of vegetative apical meristems, and later expanding to the whole inflorescence meristem, as well as primordia and organs of third and fourth floral whorls. This specific expression pattern suggests a role in the organization of reproductive meristems, as well as during flower organ development.     

Type XXVI collagen,a new member of the collagen family,is specifically expressed in the testis and ovary

HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen.