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PubMed1995

Differential display and integrin alpha 6 messenger RNA overexpression in hepatocellular carcinoma.

Our aim was to isolate potentially important differentially expressed gene products from paired human hepatocellular carcinoma (HCC) and normal liver samples using the differential messenger RNA (mRNA) display technique. Total RNA samples were reverse transcribed with anchoring oligonucleotide primers and then amplified by the polymerase chain reaction (PCR) with additional upstream random primers. Differentially expressed complementary DNA (cDNA) products were subsequently used as probes in Northern blot analysis. One such cDNA product, present in tumor but absent in normal displays, showed identity with the adhesion molecule integrin alpha 6. In Northern blot of 16 HCC pairs, the approximately 5.5 kb signal of integrin alpha 6 mRNA was overexpressed in seven tumors, with a weak signal in the normal livers. For those patients with versus without integrin alpha 6 mRNA overexpression: (1) grade III (or IV) histology was noted in seven of seven versus three of nine tumors, respectively (P = .03); (2) tumor recurrence or death (at mean follow-up of 18 months) was noted in six of seven versus three of eight patients, respectively (P = .17). Similar results were obtained using semiquantitative PCR co-amplification with glyceraldehyde 3-phosphate dehydrogenase as a control; approximately 50% of the tumors had stronger integrin alpha 6 bands than their paired normals. Both A and B variants of integrin alpha 6 mRNA were detectable in the tumor and normal liver samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed2007

Regulation of the UGT1A1 bilirubin-conjugating pathway: role of a new splicing event at the UGT1A locus.

UNLABELLED: UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3' end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99-amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that isoform 2 is differentially expressed in liver, kidney, colon, and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions. CONCLUSION: Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway.

Alternative Splicing↗

PubMed1997

A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome.

The human Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of non-bile salt organic anions. A highly similar phenotype has been described for a mutant Wistar rat strain, the transport-deficient (TR-) rat, which is defective in the canalicular multispecific organic anion transporter (cmoat). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The complementary DNA (cDNA) encoding rat cmoat has recently been cloned, and the mutation underlying the defect in TR- rats has been identified. In the present study, we have isolated the human homologue of rat cmoat, human cMOAT, and analyzed the corresponding cDNA from fibroblasts of a DJS patient for mutations. Our results show that a mutation in this gene is the cause of DJS.

Amino Acid Sequence↗

PubMed1999

The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells.

A particular point mutation of the tumor suppressor gene p53, namely a G-->T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a human p53 complementary DNA mutated at the third base of codon 249 and analyzed the consequences of this gene transfer on the growth properties of this cell line. HACL-1 cells transfected with mutant p53 showed no increase in their life-span (when compared with HACL-1 cells transfected with the antibiotic resistance gene alone) and did not grow in soft agar, whereas transfection of wild-type p53 into HACL-1 cells led to a proliferation stop. Thus, these results strongly support the view that the mutation at codon 249 of the p53 gene may serve as a fingerprint for aflatoxin B1-induced hepatocellular carcinomas, but is not, by itself, sufficient to immortalize human liver cells.

Base Sequence↗

PubMedDate not supplied

Novel development-related alternative splices in human testis identified by cDNA microarrays.

Alternative splicing of premessenger RNA is an important regulatory mechanism that increases the diversity of proteins transcribed from a single gene. This is particularly important in the testis because germ cell expansion and differentiation require many cellular changes and regulatory steps. To investigate novel development-related alternative splicings in the human testis, complementary DNA microarray studies were conducted with the use of probes from human fetal testes, adult testes, and human spermatozoa. Of a total of 386 Unigene clusters found to be related to the development of the testis, 67 clusters showed a total of 74 novel alternative spliceoforms. Developmental stage-dependent expression was also performed for a novel Unigene, NYD-SP20 (Hs.351068), which had 4 possible novel spliceoforms and another Unigene, CRISP2 (cysteine-rich secretory protein 2, Hs.2042), which had 3 possible novel spliceoforms. These results indicate that alternative splicing plays an important role in the complicated processes of testis development and spermatogenesis.

Adult↗

PubMed1995

Molecular cloning and expression of mouse mg(2+)-dependent protein phosphatase beta-4 (type 2C beta-4).

A full-length complementary DNA (cDNA) clone (pTK-3) encoding an isoform of Mg(2+)-dependent protein phosphatase beta (MPP beta-4) was isolated for the first time from a mouse melanocyte cDNA library. It was strongly suggested that the mRNA corresponding to the pTK-3 insert was a splicing variant of a single pre-mRNA that also encodes MPP beta-1 and -2 (T. Terasawa, T. Kobayashi, T. Murakami, M. Ohnishi, S. Kato, O. Tanaka, H. Kondo, H. Yamamoto, T. Takeuchi, and S. Tamura, 1993, Arch. Biochem. Biophys. 307, 342-349). The amino acid sequence of MPP beta-4 differed from those of MPP beta-1 and -2 only at the carboxyl terminal region. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) revealed that MPP beta-4 mRNA was expressed only in testis and intestine and not in other mouse tissues tested. Specific expression of the mRNA signals of two other isoforms of MPP beta, MPP beta-3 and -5 (a novel isoform), in testis and intestine was also demonstrated by the RT-PCR. The carboxyl terminal region of MPP beta-5 was found to have a chimera structure composed of part of MPP beta-1 and part of MPP beta-3. The recombinant MPP beta-3 and -4 and the putative MPP beta-5 expressed in Escherichia coli cells exhibited Mg(2+)-dependent and okadaic acid-insensitive protein phosphatase activities. It was demonstrated that the mRNA expression levels of MPP beta-3, -4, and -5 alter according to the maturation of mouse testis. These results suggest that the complex structure of MPP beta isoforms and their tissue- and developmental stage-specific expression reflect the variety of their physiological functions.

Alternative Splicing↗

PubMed1994

Cloning and expression of a human glutamate transporter.

A complementary DNA clone was isolated from human brain that encodes a glutamate transporter. Injection of RNA transcribed from this cDNA into Xenopus oocytes resulted in expression of a transport activity with a high affinity for glutamate (Km = 78.4 microM) and a dependency on external Na+. The cDNA sequence predicts a protein of 542 amino acids that is highly homologous to the congeneric proteins from rat and mouse brains. RNA blotting analyses revealed the expression both in the brain and in peripheral tissues.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of the human fertilin beta subunit.

Fertilin, a heterodimeric sperm surface protein, first identified in guinea pig, has been shown to play an important role in sperm-egg interactions. We report here the complementary DNA and deduced amino acid sequence of human fertilin beta. The human fertilin beta shares significant sequence homology with mouse, guinea pig and monkey fertilin beta and also exhibits similar structural organization. Of particular interest, the mature guinea pig fertilin beta contains an amino-terminal 90 amino acid disintegrin domain. It has been suggested that the integrin recognition sequence (TDE) of the guinea pig fertilin beta disintegrin domain mediates sperm-egg binding. The amino acid sequence at this position in human fertilin beta differs from the mouse, guinea pig and monkey sequence (mouse-QDE; human-FEE; monkey-FDE).

ADAM Proteins↗

PubMed1997

Molecular cloning and characterization of a putative neural calcium channel alpha1-subunit from squid optic lobe.

The complete amino acid sequence of a putative calcium channel alpha1-subunit, SQCC1, from the optic lobe of the squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The open reading frame encodes 2206 amino acids, which corresponds to a molecular weight of 251,451. The deduced amino acid sequence shares general structural features with the other voltage-dependent calcium channels; it consists of four repeated units of homology. Each motif has five hydrophobic segments and one positively charged segment. The transcriptional products were detected in all nervous systems examined; optic lobe, cerebral ganglia and giant stellate ganglia. However, it was not detected in the mantle muscle, heart and stomach, indicating SQCC1 is a calcium channel alpha1-subunit specific for squid nervous system. SQCC1 is more closely related in its amino acid sequence patterns to dihydropyridine-insensitive calcium channels rather than dihydropyridine-sensitive ones.

Amino Acid Sequence↗

PubMed1997

Sequence of a cDNA clone encoding a novel somatolactin in goldfish, Carassius auratus.

As a first step towards the development of a sensitive ribonuclease protection assay to study the regulation of somatolactin (SL) mRNA expression in pituitary cells of goldfish, we have isolated a complementary DNA (cDNA) clone encoding precursor sequence of SL from a cDNA library prepared from goldfish pituitary poly(A)+ RNA. The 843-bp goldfish SL (gfSL) cDNA has an open reading frame of 693 nucleotides with two possible start codons of AUG. Amino acid sequence alignment revealed that gfSL has the characteristics of four conserved domains (A, B, C and D) common to all SLs with the domain B being the most conserved region among all the characterized SLs. Similar to other teleost SLs, this gfSL is similarly related but clearly distinct from growth hormone and prolactin of goldfish and other teleosts. However, unlike most other known teleost SLs which have more than 70% amino acid sequence identity to each other, the overall amino acid sequence identity of this novel gfSL with other previously characterized SLs ranges from only 36% to 51%. Moreover, this gfSL contains only six cysteine residues, rather than seven in most other SLs, in conserved positions. Northern blot analysis revealed a single gfSL mRNA transcript of approximately 1 kb in the pituitaries of both sexually regressed and maturing male and female goldfish.

Amino Acid Sequence↗

PubMed2000

Cloning and expression of mouse deafness dystonia peptide 1 cDNA.

Complementary DNA of mouse deafness dystonia peptide 1 (DDP1) was isolated from adipocyte cDNA library and expressed in mammalian cells. The sequence shares homology of 92 and 97% on the nucleic acid and the amino acid levels with human DDP1. In comparison to mouse Bruton's tyrosine kinase (Btk) locus, the coding region spans 2 exons and the splice point is the same as human DDP1. Northern blot analysis suggests that mouse DDP1 expresses ubiquitously. In vitro transcription/translation study showed that the cDNA of mouse DDP1 codes about 11 kDa peptide. DDP1 tagged with FLAG localized in mitochondria and cytoplasm of COS7 cells. P19 embryonal carcinoma cells transfected with anti sense DDP1 cDNA were frequently dead after subculture and all the survivals expressed endogenous DDP1 mRNA. Therefore, mouse DDP1 may play an important role to survive in contrast to Tim8p, a yeast homologue, which was unnecessary in yeast.

Amino Acid Sequence↗

PubMed2001

Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family.

Using a human placenta cDNA library, we cloned a novel member belonging to the scavenger receptor family. Complementary DNA of this clone encodes a type II transmembranous glycoprotein containing a collagen-like domain, which are typical structural characteristics of scavenger receptor class A. This protein also contains a C-type lectin/carbohydrate recognition domain (C-type CRD) located at the C-terminus. We designated this as Scavenger Receptor with C-type Lectin (SRCL) type I. We also isolated human SRCL type II, which lacks the C-type CRD. Northern blot analysis revealed that hSRCL type I and type II mRNAs are abundantly expressed in adult human tissues. When hSRCL type I and type II were expressed in CHO-K1 cells, they were localized in the plasma membrane forming clusters on the surface. Ligand-binding studies of CHO-K1 cells expressing hSRCL type I and type II demonstrated their specific binding capacity in Escherichia coli and Staphylococcus aureus. These results indicate that hSRCL is a novel bacteria-binding receptor containing a C-type CRD and this receptor may play an important role in host defense.

Amino Acid Sequence↗

PubMed2001

Trypanosoma evansi: cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat1.2.

A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera frugiperda [corrected] insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.

Amino Acid Sequence↗

PubMed1997

Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa.

In the Neurospora Genome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungus Neurospora crassa are being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).

Amino Acid Sequence↗

PubMed1998

Cloning of complementary deoxyribonucleic acid for the follicle-stimulating hormone-beta subunit in the Japanese quail.

Complementary DNA clones encoding the luteinizing hormone-beta and common alpha subunits have been isolated not only in mammals but also in some nonmammalian tetrapod vertebrates. However, cloning of cDNA encoding the follicle-stimulating hormone (FSH)-beta subunit is limited to mammals, and no clone for the FSH-beta subunit has been isolated for nonmammalian vertebrates. We report here the isolation and characterization of cDNA encoding the FSH-beta subunit precursor molecule in the Japanese quail from a cDNA library of the pituitary gland of this bird. As the hybridization probe for the screening, we used a cDNA clone prepared by the polymerase chain reaction (PCR) with primers designed from known nucleotide sequence data of cDNA for FSH-beta subunit precursors of mammals. The FSH-beta subunit precursor cDNA we isolated was longer than any other FSH-beta subunit precursor cDNAs previously reported. This is due to the extraordinarily long 3'-untranslated region (2135 bp). This region is extremely rich in consensus sequences reported to cause instability of mRNA, suggesting that mRNA for the FSH-beta subunit, especially that of the Japanese quail, is unstable. Northern blot analysis of mRNA for the FSH-beta subunit revealed that the pituitary content of mRNA in the nonbreeding season was about 1/5 to 1/10 that in the breeding season in male Japanese quail kept in an outdoor cage. The profile of the seasonal change in FSH-beta subunit mRNA in the quail pituitary gland was similar to that of the seasonal change in the concentration of FSH in plasma reported previously. The deduced amino acid sequence of the mature protein region showed that the quail FSH-beta subunit is more similar to the opossum FSH-beta subunit than to the other mammalian FSH-beta subunits.

Amino Acid Sequence↗

PubMed2000

Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay.

Complementary DNA (cDNA) encoding newt (Cynops pyrrhogaster) growth hormone (nGH) was cloned from a cDNA library constructed from mRNAs of newt pituitary glands and was expressed in Escherichia coli. Based on Northern blot analysis using the cDNA as a probe, the nGH mRNA was estimated to be 940 bases in length. The recombinant nGH (nGHr) had a molecular mass of 22 kDa as determined by SDS-PAGE and possessed considerable bioactivity as determined in a Xenopus cartilage assay. Using the nGHr, we produced a polyclonal antibody against nGHr. Western blot analysis of newt anterior pituitary gland homogenates revealed that this antiserum specifically detected a single 22-kDa band, and histological studies of newt pituitary gland sections showed that the cells that reacted immunologically by the anti-nGHr antiserum corresponded to those stained by an antiserum against rat GH. A radioimmunoassay (RIA) that is specific and sensitive for nGH was developed, employing the antiserum thus produced. The sensitivity of the RIA was 57 +/- 7 pg/100 microl assay buffer. Interassay and intraassay coefficients of variation were 1.22 and 2.70%, respectively. Serial dilutions of plasma and pituitary homogenate of C. pyrrhogaster yielded dose-response curves that were parallel to the standard curve. Plasma from hypophysectomized newts showed no cross-reactivity. Moreover, displacement curves obtained using pituitary homogenates of the sword-tailed newt (C. ensicauda) and the crested newt (Triturus carnifex) were also parallel to the standard curve. Mammalian and frog GHs and prolactins (PRLs), as well as newt PRL, showed no inhibition of binding, even at relatively high doses, in this RIA. The RIA was used to measure GH released from newt pituitaries in vitro. Enhancement of GH release by 10(-7) M thyrotropin-releasing hormone was observed in cultures of newt pituitaries.

Amino Acid Sequence↗

PubMed2000

Novel transcripts of the estrogen receptor alpha gene in channel catfish.

Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERalpha cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERalpha variants of different sizes. Relative to the catfish ERalpha (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERalpha (36 residues longer) and Short-ERalpha (389 residues shorter). The 5'-end of Long-ERalpha cDNA is identical to that of Medium-ERalpha but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERalpha binds estrogen with high affinity (K(d) = 3. 4 nM), similar to that previously reported for Medium-ERalpha but lower than reported for catfish ERbeta. Short-ERalpha cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERalpha RNAs that include the size range of the ERalpha cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERalpha cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERalpha antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERalpha antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERalpha mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may encode functional ERalpha or related proteins that modulate ERalpha or ERbeta activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may be to participate in the regulation of ER gene expression.

3' Untranslated Regions↗

PubMed2001

Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain.

Two different precursor genes encoding the decapeptide salmon GnRH (sGnRH) are present in most salmonid species. In rainbow trout, a precedent Southern blot study revealed the existence of two different sGnRH genes and, recently, two different genes and their complementary DNAs that encode the identical peptide sGnRH were isolated from ovary and testis. Our study confirms the existence of two different mRNAs encoding sGnRH (sGnRH mRNA-I and sGnRH mRNA-II) in the brain of rainbow trout and, for the first time, full-length complementary DNA sequences are given. Central and peripheral distributions of the two messengers are described and seem to indicate different regulation of their expression. sGnRH mRNA-I is found essentially in the olfactory bulbs and telencephalon, whereas sGnRH mRNA-II is more widely expressed in the brain. Our observations allow speculation on the respective roles of two genes encoding the same decapeptide.

Amino Acid Sequence↗