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At least 289 records · Page 16Linked to original sources
PubMed2006

Identification of Arabidopsis thaliana variants with differential glyphosate responses.

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.

Arabidopsis↗
PubMed2002

Identification of a splice variant of mouse caveolin-2 mRNA encoding an isoform lacking the C-terminal domain.

We identified a splice variant of mouse caveolin-2 mRNA having an intronic sequence in place of the third exon (Deltaex3). The entire sequence of full-length (FL) and Deltaex3 caveolin-2 mRNA was determined; their sizes were 2490 and 973 bp, respectively. The Deltaex3 mRNA encoded a putative isoform lacking the C-terminal 49 amino acids of the authentic caveolin-2. The expression level of Deltaex3 was lower than that of FL mRNA, but considerable in some culture cells and tissues. The isoform lacking the C-terminus localized to the endoplasmic reticulum, while the authentic caveolin-2 was distributed to the Golgi and the plasma membrane along with caveolin-1. The result confirmed the necessity of the C-terminal domain of caveolin-2 for the caveolar localization, and showed the existence of a novel caveolin-2 isoform, which is not recruited to caveolae even in the presence of caveolin-1.

3T3 Cells↗
PubMed2003

Identification of novel splice variants of ARNT and ARNT2 in the rat.

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.

Alternative Splicing↗
PubMed1983

Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type).

Structural studies on an amyloid fibril protein of 14 K daltons (AFj(INO] isolated from a Japanese patient who suffered from familial amyloidotic polyneuropathy were carried out to unambiguously identify its difference from normal human serum prealbumin. Sequence analyses performed by comparing peptide maps prepared from cyanogen bromide fragments and tryptic peptides of purified RCM-amyloid protein with those from RCM-prealbumin indicate that only a valine residue at position 30 in prealbumin is replaced by a methionine residue. Furthermore, it was also proved that AFj(INO) consists of four components; the prealbumin variant and its three related proteins, which are derived by successively accumulated deletion of the N-terminal three amino acid residues (Gly1, Pro2 and Thr3) from the prealbumin variant.

Amino Acid Sequence↗
PubMed1984

Identification of a prealbumin variant in the serum of a Japanese patient with familial amyloidotic polyneuropathy.

Serum prealbumin isolated from a Japanese patient with familial amyloidotic polyneuropathy (FAP) has been found to consist of a mixture of normal prealbumin and a prealbumin variant which contains a methionine for valine substitution at position 30. The prealbumin variant in the serum is identical to the prealbumin variant derived from amyloid fibrils of a Japanese FAP patient. FAP likely results from the deposition of abnormal serum prealbumin in various organs as amyloid fibrils.

Adult↗
PubMed2000

Identification of alternative splicing variants of the beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II with different activities.

The beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II (beta CaMKII) was identified by searching through an expressed sequence tag database and rapid amplification of cDNA 5'-ends and was assigned to chromosome 7. Reverse transcription-polymerase chain reaction and sequencing analysis identified at least five alternative splicing variants of beta CaMKII (beta, beta6, betae, beta'e, and beta7) in brain and two of them (beta6 and beta7) were first detected in any species. When expressed in HEK 293 cells, the Ca(2+)/calmodulin-dependent kinase activity of beta7, the shortest variant, was much lower than that of either beta (the longest one) or betae (the medium one), suggesting possible regulation of beta CaMKII activity by alternative splicing.

Alternative Splicing↗
PubMed1998

Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor: its regional distribution and developmental expression in the rat brain.

A longer variant of rat mRNA encoding the NR3 subunit of the NMDA receptor has been identified. It contains a 60-bp insertion at the nucleotide position 3007 in the intracellular domain of the C-terminal of the previously cloned variant. Therefore, the NR3 mRNA exists in at least two variants--with the insert (NR3-long; NR3-l) and without the insert (NR3-short; NR3-s). The NR3-l variant is expressed throughout the adult rat brain. Moreover, this variant predominates in the occipital and entorhinal cortices, thalamus and cerebellum. Analysis of NR3-l development indicates that it is regulated in a region-specific manner.

Animals↗
PubMed1999

Identification of a splice variant of neutrophil collagenase (MMP-8).

We have identified a splice variant of human neutrophil collagenase (MMP-8) transcript (MMP-8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP-8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP-8, the translation product of MMP-8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP-8alt protein may lead to uncover novel roles it plays in cellular physiology.

Alternative Splicing↗
PubMed1999

Identification of a splice variant mRNA of p40phox, an NADPH oxidase component of phagocytes.

Northern blot analysis using p40phox cDNA probe revealed that two sizes of p40phox mRNAs were expressed in human promyelocytic HL-60 and bone marrow cells. To characterize these mRNAs, we performed reverse transcription using total RNA from HL-60 cells, and amplified the coding region of p40phox by polymerase chain reaction with oligonucleotide primers. Two cDNA fragments with different sizes were isolated. One was identical to a known p40phox cDNA (1054 bp) which encoded a protein of 339 residues (39,031 Da) with a calculated pI of 6.5. The other cDNA (1299 bp) contained an additional 245 bp intron 8 sequence in the open reading frame and encoded a protein of 348 residues (39,000 Da) with a calculated pI of 9.3. N-terminal 253 residues were identical between p40phox and the variant protein, whereas C-terminal 254-348 residues of the variant protein shared low homology with p40phox. Interestingly, the variant protein lacked PC (Phox and Cdc24p) motif of p40phox, which is assumed to be important for the interaction with p67phox. In addition, Western blot analysis revealed that the variant protein was not detected in HL-60 cells and neutrophils. Together, these observations suggest that alternatively spliced variant mRNA of p40phox is expressed, but its protein is hardly present in myeloid cells.

Alternative Splicing↗
PubMed2002

Human oestrogen receptors: differential expression of ER alpha and beta and the identification of ER beta variants.

Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER alpha, NR3A1) and beta (ER beta, NR3A2) have been identified. ER beta mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER beta is distinct from that of ER alpha. A number of variant isoforms of the wild type beta receptor (ER beta 1), have been identified. In the human these include: (1). use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER beta 1L) or a truncated form (487aa hER beta 1s); (2). deletion of exons by alternative splicing; (3). formation of several isoforms (ER beta 2-beta 5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER beta1 as well as to three of the C terminal isoforms (beta2, beta 4 and beta 5). Using these antibodies we have found that ER beta 2, beta 4 and beta 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens. In conclusion, in addition to the differential expression of full length ER alpha and ER beta a number of ER variant isoforms have been identified. The impact of the expression of these isoforms on cell responsiveness to oestrogens may add additional complexity to the ways in which oestrogenic ligands influence cell function.

Alternative Splicing↗
PubMed2000

Identification of novel trkA variants with deletions in leucine-rich motifs of the extracellular domain.

The peripheral expression of trkA encoding for NGF receptor was investigated by RNase protection assay. A thymus-specific protected fragment was identified. Using 5' rapid amplification of cDNA ends, three different trkA fragments were characterized. The longer fragment corresponded to the classical trkA L3 transcripts while the two shorter fragments lacked sequences encoding for leucine-rich motifs of the extracellular domain of TrkA, similarly to the trkB L1 and L0 variants. RT-PCR analysis of adult rat tissues showed the expression of trkA L1 transcripts in the thymus, testis, lung and kidney but not in the central nervous system. Their combined expression with trkA L3 transcripts suggests that specific peripheral TrkA oligomers may modulate NGF binding and function in non-neuronal cells.

Amino Acid Motifs↗
PubMed1999

Identification of human cytomegalovirus variants by analysis of single strand conformation polymorphism and DNA sequencing of the envelope glycoprotein B gene region-distribution frequency in liver transplant recipients.

Single strand conformation polymorphism analysis (SSCP) of PCR-amplified DNA and subsequent DNA sequencing of human cytomegalovirus (HCMV) glycoprotein B (gB) gene were applied to identify known HCMV strains and to detect new virus variants. 61 HCMV PCR positive patients were studied out of a cohort of 410 patients after liver transplantation (LTX). SSCP was able to distinguish between strains Davis, AD169, and Towne, and in addition could identify five new virus variants (Berlin B, C, E, F, and H). Their frequency, gB and gH types were determined. Simultaneous infections with two or three strains or variants, as well as a switch from one virus to another virus were observed during long-term follow-up. No correlation between the occurrence of certain virus strains or gB types and defined clinical manifestations of HCMV infection after LTX was drawn.

Base Sequence↗
PubMed1997

Identification of a splice variant of the rat estrogen receptor beta gene.

Recently a second estrogen receptor termed estrogen receptor beta (ERbeta) has been cloned and characterized, and shown to be expressed at the highest levels in ovarian granulosa cells and prostatic epithelium. In the course of amplifying a region of the ligand-binding domain of the rat ERbeta cDNA we identified a second, larger transcript which appears to arise through differential splicing. The second isoform has 54 nucleotides inserted after position 1372 encoding 18 additional amino acids. Both isoforms are expressed at similar relative abundance in a range of tissues.

Amino Acid Sequence↗
PubMed2002

Genomic structure and identification of a truncated variant message of the mouse estrogen receptor alpha gene.

Estrogen receptor alpha (ERalpha) is a ligand-dependent transcription factor that directs the transcription of a wide number of estrogen-regulated genes. ERalpha mediates the effects of 17-beta-estradiol in both males and females, and was the first estrogen receptor identified. Despite the cloning of the mouse ERalpha cDNA over 15 years ago, the precise genomic organization of the mouse ERalpha gene has not yet been elucidated. In order to determine the structure of this gene, overlapping BAC and P1 clones containing partial genomic sequences of the mouse ERalpha cDNA were obtained from a mouse ES cell genomic library. Using standard restriction fragment analysis followed by Southern blotting, the mouse ERalpha gene was determined to be greater than 220 kb in length. The introns vary widely in size, from 1.8 to 60 kb in length. Sequencing of intron-exon boundaries shows that these boundaries are highly conserved between the human and mouse ERalpha genes. Additionally, we have identified a splice variant message of mouse ERalpha arising from a failure to properly splice at the 3' end of exon 4; the resulting message is predicted to produce a protein lacking the ligand-binding domain. Variant message was detected by RT-PCR in several tissues, including uterus, ovary, mammary gland, placenta and testis.

Alternative Splicing↗
PubMed1998

Characterization of canine photoreceptor phosducin cDNA and identification of a sequence variant in dogs with photoreceptor dysplasia.

Photoreceptor dysplasia (pd) is an autosomal recessive disease of miniature schnauzer dogs causing retinal degeneration. The disease is a homologue of retinitis pigmentosa, a group of genetically heterogeneous diseases, causing blindness in humans. A subtraction library was prepared from retinas of pd affected and age-matched normal control dogs to isolate de novo candidate genes for further examination. From the subtraction library, cDNA for phosducin (PDC), a member of the phototransduction pathway, was isolated as a transcript expressed at a higher level in the affected retina. First, the normal canine PDC cDNA was characterized to evaluate the PDC gene in the pd-affected retina. The characterized region of normal PDC cDNA spans 1258 nucleotides (nt) that include 738 nt of coding sequence predicted to encode a protein (Mr=28 209) of 245 amino acids (aa). Over the coding region, PDC shares 86-95% nt sequence identity and 90-95% identity in the deduced aa sequence with homologous mammalian sequences. A major transcript (1.9 kb) was observed only in retina by Northern analysis, but low levels of transcript were detected in brain, liver and kidney by reverse transcription and polymerase chain reaction. Retinal immunocytochemistry showed that PDC was detected only in rod photoreceptors, mainly in the inner segment and perinuclear region. By Northern blot analysis, increased PDC expression was observed in pre-degenerate affected retina relative to the age-matched normal. In pd- affected miniature schnauzer pedigree, a missense mutation was detected in codon 82 (CGA to GGA) that would create a non-conservative substitution (Arg to Gly) in close vicinity to the residue (Glu 85) which directly interacts with the betagamma-subunits of transducin. Only pd-affected dogs were found to be homozygous for the mutant allele, and none among 48 dogs tested from 20 other dog breeds had this allele, suggesting that the mutation could be causally associated with pd in miniature schnauzers. However, since some affected dogs are heterozygous for the mutant allele, and some are homozygous for the wild-type allele, this putative PDC missense mutation, if it is indeed a disease causing mutation, does not account entirely for the genetics of inherited retinal degeneration in the miniature schnauzer breed.

Amino Acid Sequence↗