Initial Functional Characterization Research Articles

Patellin1, a Novel Sec14-Like Protein, Localizes to the Cell Plate and Binds Phosphoinositides

Membrane trafficking is central to construction of the cell plate during plant cytokinesis. Consequently, a detailed understanding of the process depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles to the developing plate, as well as those that participate in its consolidation and maturation into a fully functional partition. Here we report the initial biochemical and functional characterization of patellin1 (PATL1), a novel cell-plate-associated protein that is related in sequence to proteins involved in membrane trafficking in other eukaryotes. Analysis of the Arabidopsis genome indicated that PATL1 is one of a small family of Arabidopsis proteins, characterized by a variable N-terminal domain followed by two domains found in other membrane-trafficking proteins (Sec14 and Golgi dynamics domains). Results from immunolocalization and biochemical fractionation studies suggested that PATL1 is recruited from the cytoplasm to the expanding and maturing cell plate. In vesicle-binding assays, PATL1 bound to specific phosphoinositides, important regulators of membrane trafficking, with a preference for phosphatidylinositol(5)P, phosphatidylinositol(4,5)P(2), and phosphatidylinositol(3)P. Taken together, these findings suggest a role for PATL1 in membrane-trafficking events associated with cell-plate expansion or maturation and point to the involvement of phosphoinositides in cell-plate biogenesis.

Association of estrogen receptor beta gene polymorphisms with bulimic disease in women.

In this study, we explored the potential association between estrogen receptor beta (ERbeta) and disease in a group of bulimic women. Eating disorders are much more common in females than in males, suggesting a possible role for female sex hormone signalling in the pathogenesis of these diseases. Furthermore, estrogen has been implicated in appetite regulation. The occurrence of menstrual disturbances is also increased in bulimic women. We studied 76 bulimic women and 60 controls, and found an association between two common polymorphisms in the ERbeta gene with disease in this group of bulimic women. More detailed characterisation of the ERbeta gene identified a novel variant changing the primary structure of ERbeta protein in one bulimic patient. An initial functional characterization of this variant did not reveal any differences compared to the wild-type protein. Our findings point towards a possible role of ERbeta and/or neighboring genes in the etiology of disease in bulimic patients.

Identification of the bZIP and Rta Homologues in the Genome of Rhesus Monkey Rhadinovirus

Rhesus monkey rhadinovirus (RRV) is a γ2-herpesvirus identified from the peripheral blood mononuclear cells of rhesus macaques (Macaca mulatta). Serologic studies suggested that the infections of RRV are prevalent among rhesus monkeys. RRV can be efficiently propagated and grows to a high titer in cultured rhesus monkey fibroblasts, thus providing a valuable model to study the rhadinovirus replication. By comparative genomic studies, here we describe the identification of two potential transcriptional factors encoded by RRV, designated as R-Rta and R-bZIP. Initial functional characterization of these products suggested that R-Rta is a potent transcriptional activator and R-bZIP forms homodimers in vivo. Viral homologues of R-Rta and R-bZIP, previously identified from other rhadinoviruses, have been implicated in serving as molecular switches in lytic replication.

Molecular Cloning and Functional Expression of the Potassium-Dependent Sodium–Calcium Exchanger from Human and Chicken Retinal Cone Photoreceptors

Light causes a rapid lowering of cytosolic free calcium in the outer segments of both retinal rod and cone photoreceptors. This light-induced lowering of calcium is caused by extrusion via a Na-Ca exchanger located in the rod and cone outer segment plasma membrane and plays a key role in the process of light adaptation. The Na-Ca exchanger in retinal rod outer segment was shown earlier to be a novel Na-Ca+K exchanger (NCKX), and its cDNA was obtained by molecular cloning from several mammalian species. On the other hand, the proper identity of the retinal cone Na-Ca exchanger, in terms of both functional characteristics (e.g., requirement for and transport of potassium) and molecular identity, has not yet been elucidated. Here, we report the molecular cloning, intraretinal localization by in situ hybridization, and initial functional characterization of the chicken and human cone-specific Na-Ca exchangers. In addition we report the chicken rod-specific NCKX. We identified NCKX transcripts in both human and chicken cones and observed strong potassium-dependent Na-Ca exchange activity after heterologous expression of human and chicken cone NCKX cDNAs in cultured insect cells. In situ hybridization in chicken retina showed abundant rod NCKX transcripts only in rod photoreceptors, whereas abundant cone NCKX transcripts were found in most, if not all, cone photoreceptors and also in a subpopulation of retinal ganglion cells. A detailed comparison with the previously described retinal rod and brain NCKX cDNAs is presented.

E-selectin expression and function in a unique placental trophoblast population at the fetal-maternal interface: regulation by a trophoblast-restricted transcriptional mechanism conserved between humans and mice.

Trophoblast are the earliest differentiated cells to emerge during mammalian ontogeny. Proper differentiation and maturation of trophoblast contributes to the fetal-maternal vascular interface of the mature placenta and is required for all subsequent stages of embryogenesis. Although lineage commitment and early differentiation of trophoblast have been investigated experimentally, molecular markers and regulatory mechanisms operating later in trophoblast development remain uncertain. We now report that E-selectin is expressed in a unique pattern in secondary trophoblast giant cells, trophoblast lining the central artery, and a subpopulation of labyrinthine trophoblast all located at the fetal-maternal interface of the murine placenta. These cells line vascular channels but express a unique profile of gene products not displayed by vascular endothelium. Placentae lacking E-selectin show increased trophoblast glycogen cells and fewer labyrinthine neutrophils compared with normal placentae, suggesting that recognition of E-selectin on trophoblast by counter-receptors on other cells contributes to placental development. Novel, distant first exons direct E-selectin expression in both murine and human placentae, suggesting that evolutionarily conserved and lineage-restricted transcriptional mechanisms regulate expression in homologous trophoblast populations in both species. These results define, at molecular and anatomic levels, a unique population of trophoblast located at the physiologically critical fetal-maternal vascular interface in mice. We also present initial functional characterization of E-selectin in placenta. These results support the general hypothesis that endothelial-leukocyte adhesion molecules performing specialized functions in adults may also function in development of human and murine hemochorial placentae.

The Expression of Poly(A)-binding Protein Gene Is Translationally Regulated in a Growth-dependent Fashion through a 5′-Terminal Oligopyrimidine Tract Motif

Poly(A)-binding protein (PABP) is an important regulator of gene expression that has been implicated in control of translation initiation. Here we report the isolation and the initial structural and functional characterization of the human PABP gene. Delineation of the promoter region revealed that it directs the initiation of transcription at consecutive C residues within a stretch of pyrimidines. A study of the translational behavior of the corresponding mRNA demonstrates that it is translationally repressed upon growth arrest of cultured mouse fibroblasts and translationally activated in regenerating rat liver. Furthermore, transfection experiments show that the first 32 nucleotides of PABP mRNA are sufficient to confer growth-dependent translational control on a heterologous mRNA. Substitution of the C residue at the cap site by purines abolishes the translational control of the chimeric mRNA. These features have established PABP mRNA as a new member of the terminal oligopyrimidine tract mRNA family. Members of this family are known to encode for components of the translational apparatus and to contain an oligopyrimidine tract at the 5' terminus (5'TOP). This motif mediates their translational control in a growth-dependent manner.

Molecular Cloning and Characterization of LR3, a Novel LDL Receptor Family Protein with Mitogenic Activity

We report molecular cloning and initial functional characterization of a novel member of the low density lipoprotein receptor (LDLR) gene family. The cDNA was isolated from a human osteoblast cDNA library and encoded a 1,615 amino acids protein designated as LR3. It has, in the extracellular region, a cluster of three LDLR ligand binding repeats at a juxtamembrane position and four EGF precursor homology domains separated by YWTD spacer repeats. The entire ectodomain shares the same modular organization with the middle portion of the extracellular regions of two LDLR family members, LDLR-related protein (LRP), and gp330/megalin. LR3 mRNA was expressed in most of the adult and fetal tissues examined. The highest expression level was seen in aorta. In human osteosarcoma cells examined, LR3 mRNA was highly enriched in TE85 cells, moderately expressed in MG63 cells and primary human osteoblasts, and undetectable in SaOS-2 cells. NIH 3T3 cells transfected with either full length LR3 or its ectodomain showed significantly increased proliferation, whereas transfection of intracellular domain had no proliferative effect. We predict that LR3 is a multi-functional protein with potential mitogenic activity.

Completion of the Saccharomyces cerevisiae genome sequence allows identification of KTR5, KTR6 and KTR7 and definition of the nine-membered KRE2/MNT1 mannosyltransferase gene family in this organism.

The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White.

Completion of the Saccharomyces cerevisiae Genome Sequence Allows Identification of KTR5, KTR6 and KTR7 and Definition of the Nine‐Membered KRE2MNT1 Mannosyltransferase Gene Family in this Organism

The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White. The sequences have been deposited in the GenBank data library under Accession Numbers Z71305; U39205; Z46728.©1997 John Wiley & Sons, Ltd.

The positively acting amdA gene of Aspergillus nidulans encodes a protein with two C2H2 zinc-finger motifs.

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdl66 and amdl666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles. The wild-type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880-amino-acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains. The amdA7 semi-dominant gain-of-function mutation results in a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5' region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc-finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.

The Gene for Biotin Synthase from Saccharomyces cerevisiae: Cloning, Sequencing, and Complementation of Escherichia coli Strains Lacking Biotin Synthase

Biotin synthase catalyzes the insertion of a sulfur atom between two carbon atoms of dethiobiotin to form biotin in the last step of the biotin biosynthesis pathway. In Escherichia coli, biotin synthase is coded for by bioB gene. We report here cloning, sequencing, and initial functional characterization of the yeast gene for biotin synthase in Saccharomyces cerevisiae. We have named this gene BIO2. It consists of a 355-codon open reading frame near the ZUO1 gene. Analysis of the yeast protein encoded by the BIO2 gene reveals that it shares extensive homology with biotin synthases of E. coli and Bacillus sphaericus. The yeast and the two bacterial biotin synthase proteins have similar molecular weights, amino acid compositions, and hydropathies. The plasmid pUCBIO2 containing the yeast BIO2 gene completely complements E. coli bioB − and Δ bio mutants and enables these mutants to grow on dethiobiotin. Although BIO2 is physically linked to ZUO1, which encodes the putative left-handed Z-DNA binding protein zuotin, it appears to be regulated independently from it. The yeast BIO2 and ZUO1 genes reside near ADE3 gene on chromosome VII. BIO2 is the first eukaryotic gene reported from the biotin biosynthetic pathway.

Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida

The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate. The clcR regulatory gene, which activates the clcABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate. ClcR activated the clcA promoter, which controls expression of the clcABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid. Specific binding of ClcR to the clcR-clcA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the clcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35S labeling of the protein from a T7 promoter-controlled ATG fusion construct. ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM. ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the clcA promoter. Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon.

TGF-β1 binding protein: A component of the large latent complex of TGF-β1 with multiple repeat sequences

TGF-beta occurs in a latent complex of high Mr. We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-beta 1 complex, denoted TGF-beta 1 binding protein (TGF-beta 1-BP). Most of the sequence of fibroblast TGF-beta 1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. beta-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-beta 1-BP purified from human platelets is considerably smaller than the fibroblast form (125-160 kd vs. 170-190 kd), suggesting that there is alternative splicing of the TGF-beta 1-BP gene or that TGF-beta 1-BP undergoes cell-specific proteolysis. TGF-beta 1-BP was found not to bind and inactive TGF-beta 1; its role in the latent complex is discussed.

Characterization and functional properties of tumor cell lines in accessory cell replacement assays.

This study reports the initial phenotypic and functional characterization of a series of cloned, murine, myelomonocytic tumors and their parent cell line WEHI-3, and a group of murine B lymphoma tumors. The tumor cell lines of the myelomonocytic lineage demonstrated the ability to reconstitute a macrophage-depleted, primary in vitro anti-SRBC PFC response, but only marginally enhanced an accessory cell-depleted, ova-primed, lymphocyte proliferation in vitro response. The B lymphoma tumors displayed exactly the reverse functional profile, being highly efficient in reconstitution of the proliferation response, but not supporting the SRBC PFC response. Detailed analysis of the cell surface phenotype of the various B lymphoma tumors used in this study show they display cell surface markers characteristic of normal B lymphocytes but are heterogeneous in their various stages of differential arrest. Future work will concentrate on the orchestration of Ia-mediated and soluble factor-mediated (IL 1) modalities of antigen-triggering of lymphocytes by these B lymphoma and myelomonocytic tumor cell lines.