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Query: UNIPROT:P06889 (Mol)
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Complementary DNA for the guinea pig adrenocortical estrogen sulfotransferase (EST) has been cloned and expressed. Oligonucleotides, based on amino acid sequences of the purified 34-kilodalton protein, were synthesized and used to generate a specific probe by polymerase chain reaction for screening a guinea pig adrenal cDNA library. The polymerase chain reaction rapid amplification of cDNA ends procedure was employed to obtain the 3' and 5' cDNA ends, and a full-length cDNA was constructed. The cloned cDNA consists of 1192 base pairs and encodes a protein of 296 amino acids with a calculated molecular mass of 35,161 daltons. A computer search of the protein data banks revealed significant homology with several sulfotransferases: 71% with bovine placental estrogen sulfotransferase, 52% with rat liver phenol sulfotransferase, 35% with rat liver hydroxysteroid sulfotransferase, and 36% with rat liver senescence marker protein 2. The EST cDNA was inserted into the pcDNA I eukaryotic expression vector and transfected into COS-7 cells. The successful expression of EST cDNA in COS-7 cells was ascertained by Western blot analysis using antibody generated against the protein used to obtain the original amino acid sequence. Additionally, the expressed protein was clearly functional. Only after transfection with EST cDNA was there detectable estradiol sulfotransferase activity in COS-7 cell cytosol. The expressed EST had a single pI of 6.4, whereas native guinea pig adrenocortical EST exhibits four primary charge isoforms. The majority of adrenocortical EST activity focuses as a broad bimodal band in the pH range of 6.6-6.2; additionally, three other discrete immunocross-reactive isoforms are present with pIs of 5.5, 5.4, and 5.2. Antibodies generated against each individual isoform cross-react with all the other isoforms and with the expressed protein. These isoforms were previously reported to be isomers of a pregnenolone-binding protein; however it is now evident that the isoforms and antibodies raised against them are EST specific. Under high stringency hybridization conditions, EST mRNA was only detected in the adrenal gland, where two mRNA species of 1.4 and 1.8 kilobases were evident; when low stringency conditions were used, a faint 1.4-kilobase band was also detected in the liver. Primer extension analysis revealed that the multiple mRNAs do not arise from differential transcription initiation sites, and genomic Southern blot analysis indicated that the multiple mRNAs arise from a single gene.
Mol Endocrinol 1992 Aug
PMID:Molecular cloning and expression of a full-length complementary DNA encoding the guinea pig adrenocortical estrogen sulfotransferase. 140

Estrogen sulfotransferase (EST) activity measured under optimal in vitro conditions in the 105,000 g cytosols (HSS) of homogenized intrauterine tissues (amnion, chorion, endometrium, decidua basalis and placenta) from guinea-pigs at the 50th day of gestation indicated that the highest specific activity occurred in the chorion. EST activity in the chorion increased from day 34 (early gestation) to peak around day 45 (mid-gestation), before significantly decreasing around day 50 and further declining to barely detectable levels beyond day 60 (late gestation, the onset of parturition). 17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) activity in the chorion was almost completely membrane associated. The specific activity of the 17 beta-HSD reduction reaction in the 105,000 g pellet was 2.5-fold higher at mid-gestation than at late gestation, while the specific activity of the 17 beta-HSD oxidation reaction was 1.7-fold higher at mid-gestation as compared with late gestation. When intact pieces of chorion tissue from mid- and late gestation were incubated with 5 nM [3H]estradiol (E2), approx. 80% of the recovered free estrogen was E1 (estrone). Only chorion from animals at the onset of parturition were able to produce detectable amounts of E2 from 5 nM [3H]E1. Under the same experimental conditions the ratio of estradiol sulfate (E2S) to estrone sulfate (E1S) isolated from the media and methanol washes of late gestation chorion tissue was 3-4 times greater than for the day 45 tissue.
J Steroid Biochem Mol Biol 1991 Feb
PMID:Estrogen sulfotransferase and 17 beta-hydroxysteroid dehydrogenase activities in guinea-pig chorion through gestation. 184 44

C2H2-type zinc finger genes comprise one of the largest gene families in the human genome. These proteins are involved in genetic regulation and development and are quite conserved throughout evolution. The finger domains commonly contain the small linker peptide TGEKP between some finger units. Here, we report the isolation of 133 human zinc finger cDNAs, of which 118 are novel. These clones were isolated from human brain cDNA libraries using oligonucleotide hybridization followed by expressed sequence tag (EST) analysis, sequencing from the conserved linker region using degenerate oligonucleotide primers. This directed partial sequencing approach to cDNA isolation and characterization, signature sequencing, combines the speed of EST automatic sequencing with the focus of specific cDNA family analysis. Signature sequencing minimizes the generation of less informative random EST sequences and provides a unique relative position for sequence comparison. We also show that there is an even distribution of these RNAs from this brain cDNA library, and that these cDNAs contain N-terminal domains found in other zinc finger genes. This rapid focused sequencing approach should be applicable to any family of cDNAs containing short conserved signature peptide sequences.
Hum Mol Genet 1995 Apr
PMID:Rapid isolation and characterization of 118 novel C2H2-type zinc finger cDNAs expressed in human brain. 763 19

Estrogen sulfotransferase (EST) purified from the guinea pig (gp) adrenal gland consists of multiple charge isoforms with isoelectric points (pls) ranging from 6.5 to 5.2. Four individual isoforms were isolated for use in antibody production, as well as for tryptic fragmentation analysis. The multiple charge isoforms manifested a high degree of relatedness as evidence by complete immunocross-reactivity. This relatedness was further demonstrated by peptide mapping analysis, which revealed essentially identical elution profiles for three of the isoforms, whereas the fourth most acidic isoform, although similar to the other three isoforms, demonstrated some differences. To further explore the nature of the charge isoforms, native gpEST and EST expressed by CHO-K1 cells transfected with the gpEST cDNA were subjected to additional biochemical characterization. An interesting finding was that, in addition to EST activity, gpEST bound estradiol with a high affinity [dissociation constant (Kd) 10 nM]; furthermore, the binding activity was absolutely dependent on the cofactor, adenosine-3',5'-diphosphate (3',5'-ADP). An unexpected and novel finding was that only the most basic pl 6.5 isoform was catalytically active, while estradiol-binding activity was associated with a more acidic isoform (pl 5.5-5.2); however, it has not as yet been possible to definitively assign the binding activity to a specific isoform.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Dec
PMID:Structural and functional characterization of estrogen sulfotransferase isoforms: distinct catalytic and high affinity binding activities. 770 52

A new isoform of rat liver estrogen sulfotransferase (EST), rEST-6, which is distinct from the previously reported rat EST [Demyan et al., Molec. Endocrinol. 6 (1992) 589], has been cloned, expressed, purified and characterized. A PCR procedure using oligonucleotide primers synthesized to the 5'-nontranslated and 3'-nontranslated regions of the published rEST sequence was used to isolate rEST-6 cDNA. The cloned DNA is 1000 bp in length and encodes a protein of 295 amino acids with a calculated molecular mass of 35,300 Da. rEST-6 is selectively expressed in male rats, as confirmed by Northern blot and immunoblot analyses. Northern blot analysis of male and female rat liver RNA with the rEST-6 cDNA as a probe shows a band with male RNA but not with female RNA. Similarly, immunoblot analysis of male and female rat liver cytosols with an antibody to rat EST yields a strong immunoreactive band in rat liver cytosol from male rats but not from females. Subsequent to bacterial expression and purification of rEST-6, the enzyme was analyzed kinetically and shown to sulfate estrogens but not dehydroepiandrosterone, pregnenolone, cortisol or testosterone. Maximal sulfation activity towards both beta-estradiol and estrone occurred at a concentration of 1 microM with substrate inhibition at higher concentrations. These results indicate that multiple, closely related forms of EST are present in rat liver. Analysis of the activity and regulation of these different EST enzymes is important in understanding estrogen metabolism in rats.
J Steroid Biochem Mol Biol 1995 Jan
PMID:Isolation and expression of an isoform of rat estrogen sulfotransferase. 785 71

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently we isolated four cDNAs encoding membrane-bound ligands to hek and elk [Beckman et al. (1994) EMBO J. 13, 3757-3762; Kozlosky et al. (1995) Oncogene 10, 299-306]. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. A search of GenBank revealed an expressed sequence tag (EST) with homology to the LERKs. Using this EST as a probe, we have isolated human and murine cDNAs that encode a protein which we call LERK-5. The human and murine cDNAs encode proteins of 333 and 336 amino acids, respectively, with a 97% amino acid identity; LERK-5 has an amino acid identity of 27-59% with the other reported LERKs. LERK-5 is a ligand for both elk and hek and induces receptor phosphorylation. It is expressed in adult lung and kidney and the fetal tissues heart, lung, kidney, and brain. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that LERK-5 (Eplg5) maps to the proximal region of mouse chromosome 8.
Mol Immunol 1995 Nov
PMID:Isolation of LERK-5: a ligand of the eph-related receptor tyrosine kinases. 855 44

In mice, natural resistance or susceptibility to infection with Mycobacteria, Salmonella, and Leishmania is controlled by a gene named Bcg. Bcg regulates the capacity of macrophages to limit intracellular replication of the ingested parasites, and is believed to regulate a key bactericidal mechanism of this cell. Recently, we have cloned the Bcg gene and shown that it encodes a novel macrophage-specific membrane protein designated Nramp. A routine search of the public databases for sequences homologous to Nramp identified 3 expressed sequence tags (EST) that show strong similarities to the mammalian protein. We report the identification and cloning of a full-length cDNA clone corresponding to a plant homologue (OsNramp1) of mammalian Nramp. Predicted amino acid sequence of the plant protein indicates a remarkable degree of similarity (60% homology) with its mammalian counterpart, including identical number, position, and composition of transmembrane domains, glycosylation signals, and consensus transport motif, suggesting an identical overall secondary structure and membrane organization for the two proteins. This high degree of structural similarity indicates that the two proteins may be functionally related, possibly through a common mechanism of transport. RNA hybridization studies and RT-PCR analyses indicate that OsNramp1 mRNA is expressed primarily in roots and only at very low levels in leaves/stem. DNA hybridization studies indicate that OsNramp1 is not a single gene, but rather forms part of a novel gene family which has several members in all plants tested including cereals such as rice, wheat, and corn, and also in common weed species. The striking degree of conservation between the macrophage-specific mammalian Nramp and its OsNramp1 plant homologue is discussed with respect to possible implications in the metabolism of nitrate in both organisms.
Plant Mol Biol 1995 Dec
PMID:The macrophage-specific membrane protein Nramp controlling natural resistance to infections in mice has homologues expressed in the root system of plants. 861 17

Dystrophin is the protein product which is absent in Duchenne muscular dystrophy (DMD). In mammalian skeletal muscle, dystrophin is found in association with several integral and peripheral membrane proteins, forming a complex known as the dystrophin glycoprotein complex (DGC). In an expressed sequence tag (EST) database search to identify new dystrophin related genes, we isolated EST00891 which showed 57% homology to the cysteine-rich domain of dystrophin and localized to 18q12.1-12.2. This EST is also highly homologous (90%) to the Torpedo californica post-synaptic 87 kDa phosphoprotein. Screening human adult brain and skeletal muscle cDNA libraries with this EST resulted in cloning multiple cDNAs which encode several splice forms all homologous to the C-terminal domain of dystrophin. The largest open reading frame isolated shows 94% homology (86% identity) to the Torpedo 87 kDa protein and 50% homology to the cysteine-rich and carboxy-terminal domains of dystrophin. The other cDNAs isolated encode smaller splice forms of this gene which we have named dystrobrevin. The tissue distribution of dystrobrevin mRNA shows five distinct transcripts which are preferentially expressed between different tissues. In addition, antibodies against either the Torpedo 87 kDa protein or human dystrobrevin demonstrate that at least three of the splice forms are translated as proteins in human brain tissue extracts.
Hum Mol Genet 1996 Apr
PMID:Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane. 884 41

hnifU, a gene exhibiting similarity to nifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins from Haemophilus influenzae and Saccharomyces cerevisiae, respectively, and 40-44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63-77%) than to the diazatrophic NifU proteins (40-48%). Further, the NifU-like proteins of non-nitrogen-fixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans and H. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins.
J Mol Evol 1996 Nov
PMID:A modular domain of NifU, a nitrogen fixation cluster protein, is highly conserved in evolution. 887 67

As an approach to characterizing all human ATP-binding cassette (ABC) superfamily genes, a search of the human expressed sequence tag (EST) database was performed using sequences from known ABC genes. A total of 105 clones, containing sequences of potential ABC genes, were identified, representing 21 distinct genes. This brings the total number of characterized human ABC genes from 12 to 33. The new ABC genes were mapped by PCR on somatic cell and radiation hybrid panels and yeast artificial chromosomes (YACs). The genes are located on human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17 and X; at locations distinct from previously mapped members of the superfamily. The characterized genes display extensive diversity in sequence and expression pattern and this information was utilized to determine potential structural, functional and evolutionary relationships to previously characterized members of the ABC superfamily.
Hum Mol Genet 1996 Oct
PMID:Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database. 889 2


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