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Results for “Protein Isoforms”

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PubMed1999

Functional analysis of activation and repression domains of the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNT) protein isoforms.

The aryl hydrocarbon receptor nuclear translocator (ARNT) protein is involved in many signaling pathways. Rainbow trout express isoforms of ARNT protein that are divergent in their C-terminal domains due to alternative RNA splicing. Rainbow trout ARNT(b) (rtARNT(b)) contains a C-terminal domain rich in glutamine and asparagine (QN), whereas the C-terminal domain of rtARNT(a) is rich in proline, serine, and threonine (PST). rtARNT(b) functions positively in AH receptor-mediated signaling, whereas rtARNT(a) functions negatively. Studies were performed to understand how changes in the C-terminal domains of the two rtARNT isoforms affect function. Deletion of the QN-rich C-terminal domain of rtARNT(b) did not affect function in aryl hydrocarbon receptor (AHR)-mediated signaling, whereas deletion of the PST-rich domain of rtARNT(a) restored function. Expression of the PST-rich domain on truncated rtARNT(b) or mouse ARNT (mARNT) reduced function of this protein by 50-80%. Gel shift assays revealed that the PST-rich domain affected AHR-mediated signaling by inhibiting DNA binding of the AHR*ARNT heterodimer. Gal4 transactivation assays revealed a potent transactivation domain in the QN-rich domain of rtARNT(b). In contrast, Gal4 proteins containing the PST-rich domain of rtARNT(a) did not transactivate because the proteins did not bind to DNA. Secondary structure analysis of the PST-rich domain revealed hydrophilic and hydrophobic regions. Truncation of the hydrophobic domain that spanned the final 20-40 amino acids of the rtARNT(a) restored function to the protein, suggesting that repressor function was related to protein misfolding or masking of the basic DNA binding domain. Functional diversity within the C-terminal domain is consistent with other negatively acting transcription factors and illustrates a common biological theme.

Animals↗

PubMed1999

Identification of a new exon in the myelin proteolipid protein gene encoding novel protein isoforms that are restricted to the somata of oligodendrocytes and neurons.

The myelin proteolipid protein (PLP) gene (i.e., the PLP/DM20 gene) has been of some interest because of its role in certain human demyelinating diseases, such as Pelizaeus-Merzbacher disease. A substantial amount of evidence, including neuronal pathology in knock-out and transgenic animals, suggests the gene also has functions unrelated to myelin structure, but the products of the gene responsible for these putative functions have not yet been identified. Here we report the identification of a new exon of the PLP/DM20 gene and at least two new products of the gene that contain this exon. The new exon, located between exons 1 and 2, is spliced into PLP and DM20 mRNAs creating a new translation initiation site that generates PLP and DM20 proteins with a 12 amino acid leader sequence. This leader sequence appears to target these proteins to a different cellular compartment within the cell bodies of oligodendrocytes and away from the myelin membranes. Furthermore, these new products are also expressed in a number of neuronal populations within the postnatal mouse brain, including the cerebellum, hippocampus, and olfactory system. We term these products somal-restricted PLP and DM20 proteins to distinguish them from the classic PLP and DM20 proteolipids. They represent putative candidates for some of the nonmyelin-related functions of the PLP/DM20 gene.

Aging↗

PubMed2004

Ribosome-inactivating activity and cDNA cloning of antiviral protein isoforms of Chenopodium album.

We have characterized a novel type I ribosome-inactivating protein (CAP30) from the leaves of Chenopodium album. Purified native CAP30 depurinated the ribosomes of Chenopodium, tomato, and tobacco leaves in vitro. To further characterize this protein, cDNA clones were isolated from a leaf cDNA library using a DNA probe derived from the N-terminal amino acid sequence. Two full-length cDNA clones, CAP30A and CAP30B, were isolated. The two clones were highly homologous (91.4% identity over 280 amino acids) at the deduced amino acid level. Both contain a putative signal peptide of 25 amino acid and a conserved domain commonly found in ribosome-inactivating proteins. This suggests that CAP30 is a single-chain ribosome-inactivating protein. Expression of CAP30 mRNA peaked twice, at 12 and 72 h, after tobacco mosaic virus (TMV) infection or wounding. Transformed Escherichia coli cells expressing pre- or mature CAP had greatly reduced growth rates. These results suggest that CAP30 functions as a broad-spectrum defense-related protein with both antiviral and anti-microbial activity.

Amino Acid Sequence↗

PubMed1992

Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis.

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hlousek-Radojcić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.

Journal Article↗

PubMed2003

Generation and characterization of mice transgenic for human IL-18-binding protein isoform a.

Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-gamma (IFN-gamma)-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-gamma and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-gamma in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli.

Animals↗

PubMed1995

The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression.

The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.

Alternative Splicing↗

PubMed2002

Down but not out? A novel protein isoform of the estrogen receptor alpha is expressed in the estrogen receptor alpha knockout mouse.

The mouse knockout of the estrogen receptor alpha (ERalpha) gene, known as alphaERKO, has been extensively used for several years to study the role and function of ERalpha. Residual estradiol binding capacity in uterine tissue of 5-10% raised doubts if this knockout is a genuine null mutation of ERalpha. Although alternatively spliced ERalpha mRNA variants in the alphaERKO mouse were reported previously, the corresponding protein isoforms have not been detected to date. Here we show that a variant ERalpha protein, 61 kDa in size, is expressed in the uterine tissue of alphaERKO mice as a result of an alternative splicing. The transactivation capability of this protein is cell dependent and can be as high as 75% of the wild type ERalpha.

3T3 Cells↗

PubMed1995

The ovarian tumor protein isoforms of Drosophila melanogaster exhibit differences in function, expression, and localization.

The Drosophila melanogaster ovarian tumor (otu) gene, required for normal proliferation and differentiation of the female germ-line, encodes two cytoplasmic protein isoforms, 98 and 104 kDa. Mutants with defects in this gene are typically grouped into one of three phenotypic classes: quiescent (germ cells do not proliferate), oncogenic or tumorous (germ-line cells proliferate uncontrollably), and differentiated (germ-line cells initiate but do not complete differentiation). Analysis of transformants expressing only one of the otu isoforms showed that the 104-kDa isoform (otu-104) can rescue all classes of otu mutants, whereas only differentiated mutants are rescued to a significant extent by the 98-kDa isoform (otu-98). Western analysis of protein extracts prepared from ovaries of various developmental stages indicated that otu-104 predominates in predifferentiated stages, while otu-98 is prevalent in differentiated egg chambers. Immunolocalization experiments demonstrated that otu protein is present in the cytoplasm of oogonial stem cells that populate third instar larvae and in all germ-line-derived cells until late in oogenesis. In stage 10 egg chambers, otu protein shifts to the subcortical region of nurse cells. This type of analysis also showed that upon formation of a 16-cell syncytium otu-104, but not otu-98, preferentially accumulates in the developing oocyte cytoplasm. The otu mutant protein does not show this pattern of enhanced accumulation, nor does it occur in ovaries of egalitarian and Bicaudal-D mutants, which are defective in oocyte determination. Thus, these studies indicate that the 104-kDa isoform is required for normal proliferation of female germline cells and perhaps for oocyte differentiation. The 98-kDa isoform appears to be dispensable but can provide an otu function needed for the completion of oocyte maturation.

Animals↗

PubMed2002

Argyrophilic grain disease and Alzheimer's disease are distinguished by their different distribution of tau protein isoforms.

Prominent neuronal and glial tau filamentous inclusions are hallmarks of neurodegenerative tauopathies, among them Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and argyrophilic grain disease (AgD). AgD is a late onset dementia in which pathologically aggregated tau proteins are found in limbic structures in the shape of distinct argyrophilic grains and coiled bodies. Until now tau protein deposits in AgD have not been assessed biochemically. We therefore decided to investigate the electrophoretic profile of pathological tau protein as well as the tau protein isoform composition of filamentous inclusions in AgD cases. A distinct pathological tau doublet at 64 and 69 kDa and a minor 74-kDa band was obtained in two AgD cases with only very mild concomitant AD pathology (Braak stage I), while in two AgD cases with moderate AD pathology (Braak stage II and III, respectively), an additional minor band at 60 kDa was detected. Thus, the pathological tau profile (PTP) in pure AgD cases differs from both the PTPs in AD (tau triplet at 60, 64 and 69 kDa, minor band at 74 kDa) and PiD (major tau doublet at 60 and 64 kDa, minor band at 69 kDa) but not from those in PSP and CBD. Using a two-dimensional gel electrophoresis approach anti-exon 10 antiserum strongly stained the AgD doublet and the minor 74-kDa band, while anti-exon 2 and 3 antisera only faintly stained the 69- and the minor 74-kDa component, thus suggesting that pathological tau aggregates in AgD are mainly made of four-repeat (4R) tau isoforms. Furthermore, in contrast to earlier immunohistochemical studies, we now show biochemically that Ser262 indeed is phosphorylated in the PTP of AgD. Finally, expression of normal tau protein was not found to be altered in AgD. Altogether, our results demonstrate that AgD is characterized by a major tau doublet that is distinct from AD and PiD. AgD, however, shares the pathological tau doublet (64 and 69 kDa) as well as the predominance of 4R tau isoforms with CBD and PSP.

Aged↗

PubMed2003

Differential distribution of vesicle associated membrane protein isoforms in the mouse retina.

PURPOSE: Many proteins associated with synaptic vesicle exocytosis are differentially distributed among synapses in the retina and elsewhere in the central nervous system. The synapse-specific distribution of these proteins and their isoforms is thought to contribute to synapse-specific functional differences. Vesicle-associated membrane protein (VAMP, also known as synaptobrevin) is an integral synaptic vesicle membrane protein that is part of the fusion core complex needed for docking and fusing of synaptic vesicles at the synaptic active zone. Two VAMP isoforms have been identified that are considered to be synaptic, VAMP-1 and VAMP-2, however their distributions among the various synapses in the mammalian retina have not been characterized. METHODS: Single- and double-labeling immunocytochemistry was used to investigate the distribution of the synaptic VAMP isoforms, VAMP-1 and VAMP-2, in the mouse retina. RESULTS: VAMP-2 was the predominant isoform in both synaptic layers. Double-labeling studies using conventional and ribbon-synapse-specific markers showed that VAMP-2 was broadly distributed among conventional and ribbon synapses. In contrast, the distribution of VAMP-1 was very limited. In the outer retina, only weak labeling was present in photoreceptor terminals. In the inner retina, labeling for VAMP-1 was found in the dendrites, cell bodies, and axons of some ganglion cells, as demonstrated by double labeling with the ganglion cell markers, microtubule-associated protein-1 and Brn-3a. VAMP-1 labeling did not colocalize with amacrine or bipolar cell markers, nor did it colocalize with other pre-synaptic markers, suggesting that VAMP-1 is not associated directly with neurotransmitter release in the inner retina. Labeling for VAMP-1 identified a set of large ganglion cells that ramified in the mid-IPL (inner plexiform layer), suggesting that they may show ON-OFF responses. Some of these cells had cell bodies displaced to the inner nuclear layer. The dendrites of the large VAMP-1-immunoreactive ganglion cells did not co-stratify with the cholinergic plexuses of the starburst amacrine cells (labeled for choline acetyltransferase) and therefore are unlikely to show directional selectivity. However, these cells are likely to receive input from bipolar cells and a population of putative glutamatergic amacrine cells. CONCLUSIONS: VAMP-1 and VAMP-2 are differentially distributed among the synapses of the mouse retina. VAMP-2 is the predominant isoform and is widely expressed at ribbon and conventional synapses in both plexiform layers. VAMP-1 expression in the mouse retina is much more limited and is not restricted to presynaptic terminals. In the OPL, VAMP-1 is co-expressed with VAMP-2 presynaptically in photoreceptor terminals. However, VAMP-1 expression in the IPL is associated with ganglion cells and does not appear to be localized to presynaptic terminals. VAMP-1 is a specific marker for a set of large ganglion cells and displaced ganglion cells that ramify in the mid-IPL and are likely to have ON-OFF physiology.

Animals↗

PubMed2000

Stimulus- and cell-type-specific regulation of CCAAT-enhancer binding protein isoforms in glomerular mesangial cells by lipopolysaccharide and cytokines.

Binding sites for the CCAAT-enhancer binding protein (C/EBP) family are present in the promoter regions of several genes that are known to be expressed by mesangial cells (MC) during the pathogenesis of glomerular inflammatory diseases. The precise regulation of the C/EBP family by agents that are known to activate MC is, however, poorly understood. We report here the action of interleukin-1 (IL)-1 and, for the first time, lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on the C/EBP expression profile and functional DNA binding activity in primary rat MC. Both cell-type- and stimulus-specific regulation of C/EBP mRNA expression and DNA binding activity were identified, with C/EBPalpha being induced by LPS, C/EBPbeta by LPS, IL-1, TNF-alpha and C/EBPdelta by LPS, IL-1, IFN-gamma, TNF-alpha and PDGF. Such differential regulation, particularly that of C/EBPbeta, may be responsible for the mediator-specific differences in the expression of C/EBP-regulated genes in MC. Additionally, the involvement of potential post-transcriptional mechanisms in the regulation of C/EBPdelta were identified. These studies provide novel insights into the stimulus-specific regulation of gene expression during renal diseases.

Animals↗

PubMed2000

Spatio-temporal expression of the trans-acting splicing factors SF2/ASF and heterogeneous ribonuclear proteins A1/A1B in the myometrium of the pregnant human uterus: a molecular mechanism for regulating regional protein isoform expression in vivo.

Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.

Alternative Splicing↗

PubMed1996

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

BACKGROUND: Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. RESULTS: In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. CONCLUSIONS: Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

Alternative Splicing↗

PubMed2001

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain.

The formation of protease-resistant prion protein (PrP-res or PrP(Sc)) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrP(C). Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high beta-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.

Amino Acid Sequence↗

PubMed2003

Expression and properties of the recombinant murine Golli-myelin basic protein isoform J37.

A recombinant form of the murine Golli-myelin basic protein (MBP) isoform J37 (rmJ37) has been expressed in Escherichia coli and isolated to 95% purity via metal chelation and ion exchange chromatography. The protein did not aggregate lipid vesicles containing acidic phospholipids, unlike the 18.5 kDa isoform of MBP. This result is consistent with J37 having a functional role prior to the assembly of compact myelin. Circular dichroic spectroscopy showed that rmJ37 had a large proportion of random coil in aqueous solution but gained alpha-helix and beta-sheet in the presence of monosialoganglioside G(M1) and PI(4)P. Thus, like "classic" MBP, J37 is intrinsically unstructured, and its conformation depends on its environment and bound ligands. Analyses of the amino acid sequence of rmJ37 predicted an N-terminal calmodulin (CaM)-binding site. It was determined via a gel-shift assay and fluorescence spectroscopy that rmJ37 and CaM interacted in a 1:1 ratio in a Ca(2+)-dependent manner. However, the interaction was weak compared with 18.5 kDa MBP.

Animals↗

PubMed2002

Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thaliana.

To further characterize the role of biotin carboxyl carrier protein isoform 2 (BCCP2) in acetyl-coenzyme A carboxylase (ACCase) function and fatty acid biosynthesis, plants with reduced or increased expression of this protein were characterized. Analysis of 38 independent Arabidopsis lines expressing antisense BCCP2 transcript behind a constitutive promoter showed no significant phenotype, though antisense transcript was highly expressed. In developing seed, BCCP2 protein was reduced by an average of 38% resulting in a 9% average decrease in fatty acid content in mature seed. Over-expression of BCCP2 behind a seed-specific napin promoter increased the amount of holo-BCCP2 in developing seed by an average of twofold, as determined with anti-biotin antibodies. Surprisingly, the average fatty acid content of T2 seed from over-expression lines was 23% lower than wild-type seed. These plants also exhibited reduced seed setting in 18 of 20 T1 lines which was coincident with increased individual seed mass. Quantification of total BCCP2 in developing siliques using anti-BCCP2 antibodies indicated that as much as 60% of total plastidial BCCP2 was in the non-biotinylated form (apo-BCCP2). Although apo-BCCP2 was highly over-expressed in developing seed, accumulation of other ACCase subunits was unaffected. The specific activity of ACCase was up to 65% lower in developing seed of over-expression lines versus wild type. This was attributed to the assembly of biologically inactive (non-biotinylated) ACCase complexes. Consistent with ACCase exerting control over de novo fatty acid synthesis, reduced activity in developing seed resulted in lower oil content, altered fatty acid composition and reduced seed setting.

Acetyl-CoA Carboxylase↗

PubMed2005

The human AKNA gene expresses multiple transcripts and protein isoforms as a result of alternative promoter usage, splicing, and polyadenylation.

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.

Alternative Splicing↗

PubMed2003

Sry expression level and protein isoform differences play a role in abnormal testis development in C57BL/6J mice carrying certain Sry alleles.

Transfer of certain Mus domesticus-derived Y chromosomes (Sry(DOM) alleles, e.g., Sry(POS) and Sry(AKR)) onto the C57BL/6J (B6) mouse strain causes abnormal gonad development due to an aberrant interaction between the Sry(DOM) allele and the B6-derived autosomal (tda) genes. For example, B6 XY(POS) fetuses develop ovaries and ovotestes and B6 XY(AKR) fetuses have delayed testis cord development. To test whether abnormal testis development is caused by insufficient Sry(DOM) expression, two approaches were used. First, gonad development and relative Sry expression levels were examined in fetal gonads from two strains of B6 mice that contained a single M. domesticus-derived and a single M. musculus-derived Sry allele (B6-Y(POS,RIII) and B6-Y(AKR,RIII)). In both cases, presence of the M. musculus Sry(RIII) allele corrected abnormal testis development. On the B6 background, Sry(POS) was expressed at about half the level of Sry(RIII) whereas Sry(AKR) and Sry(RIII) were equally expressed. On an F(1) hybrid background, both Sry(POS) and Sry(RIII) expression increased, but Sry(POS) expression increased to a greater extent. Second, sexual development and Sry expression levels were determined in XX mice carrying a transgene expressing Sry(POS) controlled by POS-derived or MUS-derived regulatory regions. In both cases one B6 transgenic line was recovered in which XX transgenic mice developed only testicular tissue but cord development was delayed despite normal Sry transcriptional initiation and overexpression. For three transgenes where B6 XX transgenic mice developed as females, hermaphrodites, or males, the percentage of XX transgenic males increased on an F(1) background. For the one transgene examined, Sry expression increased on an F(1) background. These results support a model in which delayed testis development is caused by the presence of particular DOM SRY protein isoforms and this, combined with insufficient Sry expression, causes sex reversal. These results also indicate that at least one tda gene regulates Sry expression, possibly by directly binding to Sry regulatory regions.

Animals↗