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PubMed2000

Na+ -transport modulation induces isoform-specific expression of Na+,K+ -Atpase alpha-subunit isoforms in C2C12 skeletal muscle cell.

Changes in demands for Na+ transport alter expression of the Na+,K+ -ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the alpha2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the alpha-subunit isoforms by Na+ in the C2C12 skeletal muscle cell that expresses the alpha1 and alpha2 isoforms. Western blot analysis showed that in differentiating C2C12 muscle cell, but not in undifferentiated myoblast, veratridine, a Na+ channel activator, greatly increased expression of the alpha2 isoform; expression of alpha1 was unaltered. Because the level of alpha-actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na+ transport by tetrodotoxin again failed to alter expression of alpha1. Thus, in C2C12 skeletal muscle cell, changes in Na+ transport alters expression of the alpha2, but not the alpha1 isoform. These results differ from those observed previously in muscle cells that express only the alpha1 isoform. Because mammalian skeletal muscle expresses both the alpha1- and alpha2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.

Animals↗
PubMed1990

Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.

Isoelectric focusing of the Mr 34,000 pregnenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroid-binding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3H]pregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high- and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 proteins with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms of Mr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was approximately 10-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolone-binding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule.

Adrenal Cortex↗
PubMed2001

Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V.

Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III-VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.

Adenylyl Cyclases↗
PubMed1996

Identification of murine p120 isoforms and heterogeneous expression of p120cas isoforms in human tumor cell lines.

p120cas (CAS) is a protein tyrosine kinase substrate that associates directly with the cytoplasmic tail of the cell-cell adhesion molecule E-cadherin. CAS is thus part of a multimolecular complex that, along with other cadherin-binding proteins (catenins), mediates interactions between E-cadherin and the actin cytoskeleton. Down-regulation of E-cadherin expression and defects in catenin function have been implicated in tumor metastasis, but the role of CAS in these processes has not been addressed. Recently, the study of CAS was complicated when new anti-CAS antibodies revealed the presence of at least four putative CAS isoforms that appeared to vary in abundance between cell types. Here, we identify the four major isoforms expressed in murine fibroblasts, and we show that they are products of alternative splicing. Analysis of CAS isoforms in a variety of murine cell lines indicates that motile cells like fibroblasts and macrophages preferentially express CAS1 (i.e., CAS1A and CAS1B isoforms), and epithelial cells preferentially express CAS2 (i.e., CAS2A and CAS2B isoforms), whereas nonadherent cells (e.g., B cells, T cells, and myeloid cells) do not express detectable levels of CAS. Interestingly, CAS1 expression is dramatically up-regulated in a Src-transformed Madin-Darby canine kidney cell line, indicating that the pattern of isoform expression can be altered by cell transformation. Analysis of a variety of differentiated and metastatic human tumor cell lines reveals that CAS isoform expression in these cells is quite heterogeneous. Furthermore, several poorly differentiated cell lines fail to express particular isoforms that are typically observed in well-differentiated cell lines. These data raise the possibility that unbalanced expression of CAS isoforms in human carcinomas may influence cadherin function and contribute to malignant or metastatic cell phenotypes.

3T3 Cells↗
PubMed1999

Molecular and immunologic characterization of new isoforms of the Hevea brasiliensis latex allergen hev b 7: evidence of no cross-reactivity between hev b 7 isoforms and potato patatin and proteins from avocado and banana.

BACKGROUND: Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39% to 42% to patatins recently identified as potato allergens. The complementary DNAs encoding 2 different Hev b 7 isoforms were previously reported. OBJECTIVE: The aim of this study was to determine the sequence variation of Hev b 7 and to compare the IgE reactivity of individual isoforms in vitro and in vivo. A further objective was to evaluate possible cross-reactivities between Hev b 7 and patatins and proteins from banana and avocado. METHODS: An H brasiliensis lambda ZAP complementary DNA (cDNA) library was screened with use of a Hev b 7 cDNA probe. Four Hev b 7 isoforms were produced in recombinant form and their IgE-binding capacities were compared. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate the possible cross-reactivity between Hev b 7 and recombinant potato patatin and proteins from avocado and banana. RESULTS: Two new isoforms, S2 and D2, were identified by sequencing 32 cDNA clones with full-length coding regions. All 4 recombinant isoforms displayed esterase activity and identical IgE-binding capacities. The new isoforms S2 and D2 were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No cross-reactivity was observed between Hev b 7 isoforms and potato patatin and proteins from avocado and banana. CONCLUSIONS: All 4 recombinant Hev b 7 isoforms have equivalent IgE-binding capacity and therefore represent suitable reagents for the development of in vitro and in vivo diagnostic tests. Hev b 7, patatins, and their homologs appear not to contribute to cross-reactivity in the latex-fruit syndrome.

Adult↗
PubMed2002

Cardiac troponin T isoforms affect the Ca2+ sensitivity and inhibition of force development. Insights into the role of troponin T isoforms in the heart.

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca(2+) sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher alpha-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca(2+) sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca(2+) sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.

Amino Acid Sequence↗
PubMed1998

Tropomyosin isoforms in intestinal mucosa: production of autoantibodies to tropomyosin isoforms in ulcerative colitis.

BACKGROUND & AIMS: Autoantibodies against tropomyosins (TMs) have been reported in ulcerative colitis (UC). In this study the hTM isoforms (hTM1-5) present in intestinal epithelial cells and in smooth muscle were investigated, and the immunoreactivity against hTMs by immunoglobulin G (IgG) produced in vitro by colonic mucosal lymphocytes (LPMCs) from patients with UC, Crohn's disease (CD), and controls was examined. METHODS: TMs were extracted from colonic and jejunal epithelial cells and smooth muscle, and hTM isoforms were identified using isoform-specific monoclonal antibodies by enzyme-linked immunosorbent assay and transblot analysis. The immunoreactivity of IgG produced by colonic LPMCs was analyzed against the recombinant hTM isoforms. RESULTS: The major hTM isoforms present in colonic and jejunal epithelial cells are hTM5 and hTM4, whereas intestinal smooth muscle contains the hTM1-3 isoforms. The IgG synthesized in vitro by LPMCs from UC (n = 19) recognized hTM5 and hTM1, more significantly (P < 0.04 to <0.001) when compared with CD (n = 12) and controls (n = 17). However, IgG produced by LPMCs from CD did not show such anti-hTM reactivity. Mucosal anti-hTM IgG mainly belonged to the IgG1 subclass. CONCLUSIONS: Intestinal epithelial cells and smooth muscle have distinct hTM isoforms. Patients with UC, and not CD, show mucosal autoantibody response against hTM isoforms, particularly hTM5 and hTM1.

Adult↗
PubMed1992

Molecular cloning and expression of a new rat liver cell-CAM105 isoform. Differential phosphorylation of isoforms.

An hepatocyte cell-adhesion molecule (cell-CAM105) was recently shown to be identical with the liver plasma-membrane ecto-ATPase. This protein has structural features of the immunoglobulin superfamily and is homologous with carcinoembryonic antigen proteins. We have cloned a cDNA encoding a new form of the cell-CAM105 which is a variant of the previously isolated clone. In addition to having a shorter cytoplasmic domain, the new isoform also has substitutions clustered in the first 130 amino acids of the extracellular domain. Both of these isoforms are expressed on the surface of hepatocytes with the shorter variant being the predominant form. The previously isolated cell-CAM105 (long form) has more potential phosphorylation sites than does the new isoform (short form). Both isoforms are found to be phosphorylated after incubation with [32P]phosphate in vitro, with the long form being phosphorylated to a significantly higher extent. This observed differential phosphorylation could be one of the mechanisms for the regulation of isoform functions. Using antipeptide antibodies specific for the long form and antibodies that are reactive with both isoforms, we have shown that both isoforms are localized in the canalicular domain of hepatocytes. The sequence differences between these two isoforms suggest that they are probably derived from different genes rather than from alternative splicing.

Adenosine Triphosphatases↗
PubMed1999

Discrepant expression of membrane and soluble isoforms of Fas (CD95/APO-1) in adult T-cell leukaemia: soluble Fas isoform is an independent risk factor for prognosis.

The Fas signalling system probably plays a critical role in the natural and chemotherapeutic cell death machinery, suggesting that aberrant Fas expression is involved in growth control of tumours. The membrane isoform (mFas) is a 45 kD cell surface protein containing a single transmembrane region, and induces apoptosis in normal or tumour cells, whereas the soluble isoform (sFas) lacks the transmembrane domain due to alternative splicing of the transcript and is thought to block Fas-mediated apoptosis. To clarify the clinical roles of expression of these two Fas isoforms in adult T-cell leukaemia (ATL), we investigated the levels of the Fas isoforms in 81 patients with ATL. The expression patterns of the Fas isoforms were heterogenous, and there was no significant correlation between mFas and sFas levels: 10/81 cases were negative for mFas and had high serum sFas levels, whereas the remaining 71 cases were positive for mFas and had various levels of expression of the two Fas isoforms. Irrespective of the status of mFas expression in leukaemic cells, the mRNAs encoding these isoforms were always detectable, indicating the potential for protein translation. Although mFas expressed on freshly isolated ATL cells could iduce apoptosis in vitro, positive versus negative mFas status was not associated with any clinical aspects of ATL, whereas the sFas level was strongly correlated with clinical parameters such as serum LDH activity, tumour burden, serum soluble IL-2R level, hypercalcaemia and prognosis. These results suggest that the ratio of Fas isoforms varies, and high expression of the sFas protein and message reflects the malignant behaviour of ATL and is an independent risk factor for the prognosis.

Female↗
PubMed1997

The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species.

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted Mr of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.

Amino Acid Sequence↗
PubMed1997

Tissue-specific alternative splicing of ascidian troponin I isoforms. Redesign of a protein isoform-generating mechanism during chordate evolution.

In vertebrates, troponin I (TnI) exists as shorter and longer isoforms encoded by distinct genes expressed in skeletal and cardiac muscle, respectively. We report that the protochordate ascidian Ciona intestinalis expresses a homologous set of shorter and longer TnI isoforms in body wall muscle and heart, respectively. The heart-specific segment of the ascidian longer TnI isoform shares several sequence features with vertebrate cardiac TnI but lacks the protein kinase A phosphorylation sites implicated in sympatho-adrenal control of cardiac function. In contrast with vertebrates, the ascidian longer and shorter TnI isoforms are produced from a single gene by tissue-specific alternative RNA splicing; remarkably, the molecular mechanism of TnI isoform generation has been entirely reworked during ascidian/vertebrate evolution. Because alternative splicing is the more probable chordate ancestral condition, the long/cardiac versus short/somatic muscle pattern of TnI isoforms likely existed before the occurrence of the gene duplication events that created the vertebrate TnI gene family. Thus, gene duplication was apparently not the primary engine of isoform diversity in this aspect of TnI gene family evolution; rather, it simply provided an alternative (transcriptional) means of maintaining a previously established system of isoform diversity and tissue specificity based on alternative RNA splicing.

Alternative Splicing↗
PubMed2002

p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun. The determinant roles of the isoforms in the p38 MAPK signal specificity.

p38 MAPK pathway signaling is known to participate in cell proliferation, apoptosis, and differentiation, in a manner dependent on the cellular context. The factors that determine the specific biological response in a given cell type, however, remain largely unknown. We report opposite effects of the p38 isoforms on regulation of AP-1-dependent activities by p38 activators MAPK kinase 6 (MKK6) and/or arsenite in human breast cancer cells. The p38beta isoform increases the activation of AP-1 transcriptional activities by MKK6 and/or arsenite, whereas p38gamma/p38delta inhibits or has no effect on the stimulation. The p38beta does so by increasing the levels of phosphorylated c-Jun, whereas the p38gamma and -delta isoforms may act by regulating the c-jun transcription. AP-1-dependent processes such as vitamin D receptor gene promoter activation and cellular proliferation were similarly activated by the p38beta or inhibited by the p38gamma and/or -delta isoforms. Whereas the human breast cancer cells express all four isoforms, mouse NIH 3T3 and EMT-6 cells express only some of the p38 family members, with p38beta higher in 3T3 cells but p38delta only detected in the EMT-6 line. Consistent with the positive and negative roles of p38beta and p38delta in AP-1 regulation, MKK6 stimulates AP-1-dependent transcription in NIH 3T3 but not EMT-6 cells. In support of a role of c-Jun regulation by p38 isoforms in determining AP-1 activity, the levels of endogenous c-Jun and its phosphorylated form on p38 activation are higher in NIH 3T3 cells. These results demonstrate the contrasting activities of the different p38 isoforms in transmitting the upstream signal to AP-1 and show that the expression profile of p38 isoforms determines whether the p38 signal pathway activates or inhibits AP-1-dependent processes.

3T3 Cells↗
PubMed1999

Isoforms of the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes and ovaries: the type-1 isoform is down-regulated by fertilization and by injection of adenophostin A.

Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.

Adenosine↗
PubMed2026

Spatial isoform sequencing at single-cell resolution reveals cell-type-specific spatial isoform variability in multiple brain cell types.

Spatial long-read technologies are increasingly common but usually lack single-cell resolution. This leaves unanswered whether spatially variable isoforms reflect variability within one cell type or differences in region-specific cell-type composition. Here, we developed Spl-ISO-Seq2 (500-nm resolution) and accompanying software, Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing of >450 million barcodes versus 80,000 previously. Applying this to the adult mouse brain, we compared differential isoform abundance between known regions and spatial isoform patterns independent of predefined regions. Both identified overlapping hits, for example, Rps24 in oligodendrocytes. For known Snap25 spatial isoform variation, we show that it occurs in excitatory neurons. The region-agnostic approach also uncovered patterns missed by region-based comparisons, for example, for Ighm. Notably, many spatial isoform signals are not driven by cell-type composition alone. Finally, our software is applicable to many spatial and single-cell protocols, demonstrating reproducibility between platforms (for example, Visium HD/Stereo-seq). Overall, our experimental/analytical methods enable a submicron-resolution-isoform view and open avenues for spatial isoform disease research.

Animals↗
PubMed2000

Three subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential expression of the a3 isoform during osteoclast differentiation.

Vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme with a membrane peripheral catalytic (V(1)) and an intrinsic (V(o)) sector. We have identified three cDNA clones coding for isoforms of mouse V(o) subunit a (a1, a2, and a3). They exhibit 48-52% identity with each other and high similarity to subunit a of other species. The a1 isoform was mainly expressed in brain and liver. The a2 isoform was observed in heart and kidney in addition to brain and liver. Transcripts for the a3 isoform were strongly expressed in heart and liver. The a3 isoform was induced during osteoclast differentiation, and localized in the plasma membrane and cytoplasmic filamentous structures. In contrast to a3, the a1 isoform was constitutively expressed and localized in the cytoplasmic endomembrane compartments of the same cells. These findings suggest that the a3 isoform is a component of the plasma membrane V-ATPase essential for bone resorption.

Amino Acid Sequence↗
PubMed1993

Human fibroblast tropomyosin isoforms: characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells.

A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen lambda gt10 and lambda gt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low M(r) isoforms (hTM5a and hTM5b), and a high M(r) isoform (hTMsm alpha). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsm alpha represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues.

Amino Acid Sequence↗
PubMed1991

Organ-specific occurrence and expression of the isoforms of nonspecific lipid transfer protein in castor bean seedlings, and molecular cloning of a full-length cDNA for a cotyledon-specific isoform.

Four kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L.) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phosphatidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. PronsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗