Intron splice site PCR analysis as a tool to discriminate Dekkera bruxellensis strains

2010 ◽  
Vol 61 (1) ◽  
pp. 153-157 ◽  
Author(s):  
Ileana Vigentini ◽  
Claudia Picozzi ◽  
Roberto Foschino
Keyword(s):  
Splice Site ◽  
Pcr Analysis ◽  
Dekkera Bruxellensis ◽  
Intron Splice

scholarly journals Primary structure of rat plasma membrane Ca2+-ATPase isoform 4 and analysis of alternative splicing patterns at splice site A

1995 ◽  
Vol 306 (3) ◽  
pp. 779-785 ◽  
Author(s):  
T P Keeton ◽  
G E Shull
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Splice Site ◽  
Primary Structure ◽  
Sequence Divergence ◽  
Rat Plasma ◽  
Pcr Analysis ◽  
Rat Tissues ◽  
Mrna Tissue Distribution ◽  
Splicing Patterns

We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5′-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids.

scholarly journals Utilização do Intron Splice Site primer EI-1 na discriminação de leveduras contaminantes do processo de fermentação alcoólica

2010 ◽  
Vol 30 (3) ◽  
pp. 761-765
Author(s):  
Rodrigo Bacelar da Costa-Silva ◽  
Mario Ribeiro de Melo-Júnior ◽  
Marcos Antônio de Moraes Junior
Keyword(s):  
Splice Site ◽  
Intron Splice

scholarly journals Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

1994 ◽  
Vol 14 (5) ◽  
pp. 3426-3433 ◽  
Author(s):  
B Carr ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Splice Site ◽  
General Characteristic ◽  
Chain Gene ◽  
Splice Sites ◽  
Wild Type ◽  
Reading Frame ◽  
Intron Splice ◽  
Mature Mrna ◽  
Imprecise Excision

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.

Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4036-4036
Author(s):  
Marilyn K Parra ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Splice Site ◽  
Conflicts Of Interest ◽  
Protein Isoforms ◽  
Pcr Analysis ◽  
Acceptor Site ◽  
Intermediate Structure ◽  
Splice Acceptor ◽  
Exon 2 ◽  
Regulatory Motifs ◽  
Protein 4.1R

Abstract Abstract 4036 Poster Board III-972 The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Latency-Associated Transcript (LAT) Exon 1 Controls Herpes Simplex Virus Species-Specific Phenotypes: Reactivation in the Guinea Pig Genital Model and Neuron Subtype-Specific Latent Expression of LAT

2009 ◽  
Vol 83 (19) ◽  
pp. 10007-10015 ◽  
Author(s):  
Andrea S. Bertke ◽  
Amita Patel ◽  
Yumi Imai ◽  
Kathleen Apakupakul ◽  
Todd P. Margolis ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Guinea Pig ◽  
Splice Site ◽  
Wild Type ◽  
Intron Splice ◽  
Exon 1 ◽  
Species Specific ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2 cause similar acute infections but differ in their abilities to reactivate from trigeminal and lumbosacral dorsal root ganglia. During latency, HSV-1 and HSV-2 also preferentially express their latency-associated transcripts (LATs) in different sensory neuronal subtypes that are positive for A5 and KH10 markers, respectively. Chimeric virus studies showed that LAT region sequences influence both of these viral species-specific phenotypes. To further map the LAT region sequences responsible for these phenotypes, we constructed the chimeric virus HSV2-LAT-E1, in which exon 1 (from the LAT TATA to the intron splice site) was replaced by the corresponding sequence from HSV-1 LAT. In intravaginally infected guinea pigs, HSV2-LAT-E1 reactivated inefficiently relative to the efficiency of its rescuant and wild-type HSV-2, but it yielded similar levels of viral DNA, LAT, and ICP0 during acute and latent infection. HSV2-LAT-E1 preferentially expressed the LAT in A5+ neurons (as does HSV-1), while the chimeric viruses HSV2-LAT-P1 (LAT promoter swap) and HSV2-LAT-S1 (LAT sequence swap downstream of the promoter) exhibited neuron subtype-specific latent LAT expression phenotypes more similar to that of HSV-2 than that of HSV-1. Rescuant viruses displayed the wild-type HSV-2 phenotypes of efficient reactivation in the guinea pig genital model and a tendency to express LAT in KH10+ neurons. The region that is critical for HSV species-specific differences in latency and reactivation thus lies between the LAT TATA and the intron splice site, and minor differences in the 5′ ends of chimeric sequences in HSV2-LAT-E1 and HSV2-LAT-S1 point to sequences immediately downstream of the LAT TATA.

scholarly journals P.123 Severe DNM1 Encephalopathy with Dysmyelination due to Recurrent Splice Site Pathogenic Variant

2021 ◽  
Vol 48 (s3) ◽  
pp. S54-S54
Author(s):  
AN Sahly ◽  
E Krochmalnek ◽  
J St-Onge ◽  
M Srour ◽  
KA Myers
Keyword(s):  
Splice Site ◽  
De Novo ◽  
Subcortical White Matter ◽  
Pathogenic Variant ◽  
Pcr Analysis ◽  
Global Developmental Delay ◽  
Missense Mutations ◽  
Central Domain ◽  
Splice Site Variant ◽  
T2 Hyperintensity

Background: Patients with DNM1-encephalopathy almost exclusively have missense variants, mostly in the GTPase domain of DNM1. Delayed myelination has been reported in at least three patients with DNM1-encephalopathy, all with missense mutations in the DNM1 central domain. Only one DNM1 splice-site variant has previously been reported, and the authors questioned whether the variant accounted for all aspects of the patient’s phenotype. Methods: Case-Report. Results: Our patient had hypotonia and brief multifocal tonic seizures from age-1-month. He still has profound global developmental delay, daily seizures and microcephaly. MRI-Brain at age-21-months showed T2 hyperintensity in the bilateral periventricular and subcortical white matter; spectroscopy showed a questionable lactate peak and an elevated choline peak relative to N-acetylaspartate. Clinical gene-panel identified a heterozygous de novo pathogenic variant in intron 9 of DNM1 (c.1197-8G > A; IVS9- 8G>A). In-silico tools categorized this variant as deleterious secondary to a splicing defect. RT-PCR analysis on peripheral blood was unsuccessful as DNM1 expression is extremely low outside of the brain. Conclusions: Our patient carried the same DNM1 variant previously reported, indicating this is a recurrent pathogenic splice-site variant. The spectroscopic abnormalities suggest a possible element of demyelination in DNM1 variants of the central domain, though the mechanism remains unclear.

PCR differentiation of commercial yeast strains using intron splice site primers.

1996 ◽  
Vol 62 (12) ◽  
pp. 4514-4520 ◽  
Author(s):  
M de Barros Lopes ◽  
A Soden ◽  
P A Henschke ◽  
P Langridge
Keyword(s):  
Splice Site ◽  
Intron Splice ◽  
Yeast Strains ◽  
Commercial Yeast

326 CONSTRUCTION OF A SPLICING-DEPENDENT SELECTABLE MARKER FOR GENE TARGETTING

2011 ◽  
Vol 23 (1) ◽  
pp. 259
Author(s):  
S. Cernea ◽  
K. Wells
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Splice Site ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Selectable Marker ◽  
Stop Codon ◽  
Intron Splice ◽  
G418 Resistance ◽  
Control Plasmid

Gene targeting in mammalian cells plays a crucial role in biotechnology. These experiments are characterised by low rates of homologous recombination and high rates of random integration. Therefore, many fibroblast colonies must be screened to identify a targeting event. To dramatically reduce the survival of random integration events, we have developed a splicing-dependent selectable marker strategy by introducing a mutation in a codon-optimized G418 resistance gene (mNeo). This mutation could be corrected upon homologous recombination. Since the C-terminal region of aminoglycoside phosphotransferase (AphII, Neo/Kan resistance) participates in formation of the active site of this enzyme, we hypothesised that addition of even one amino acid at the C-terminus would render this protein non-functional. To test this hypothesis, a mutation was introduced in an E. coli AphII expression vector that converted the stop codon of AphII to tryptophan (X265W, TGA > TGGTAA). This mutation was confirmed to inactivate AphII by independently characterising the G418 and Kanamycin resistance (or lack thereof) provided by the X265W mutation. To evaluate this mutation in mammalian cells, two intronless mammalian expression vectors were constructed that differed by the presence or absence of the X265W mutation. G418 resistance was only provided by the wildtype sequence, thus confirming that X265W inactivates AphII in mammalian cells. An identical mutation was then introduced into a eukaryotic expression vector based on mNEO. Further, the sequence was extended to create a 5′ intron splice site (TGA > TGGTAAGAGTT). This region was designed to direct splicing between the first and second G residues thus removing the G in the third position of the W codon. The 3′ intron splice sites was then designed to provide an A residue as the first base of the next exon so that successful splicing would correct the mutation by recreating an appropriately positioned stop codon (TGA). To evaluate this strategy in mammalian cells, two plasmids were constructed that harbored the X265W mutation embedded at the 5′ splice site of a downstream intron. In one plasmid (pSC3-G) the first base of the downstream exon begins with a G residue resulting in inactivation of AphII. In the other plasmid (pSC2-A), the first base of the downstream exon begisn with an A residue forming a stop codon that allows for active, wildtype AphII. These plasmids were transfected into porcine fetal fibroblasts and subjected to selection with G418. A positive control plasmid and pSC2-A produced colonies that were too numerous to count. A negative control plasmid and pSC3-G produced no colonies. It can be concluded that the X265W mutation can be corrected by splicing to an exon that begins with an A residue. This splicing-dependent selectable marker may prove useful in gene targeting experiments when the site of modification is followed by an exon that begins with an A.

scholarly journals U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2647
Author(s):  
Namjeong Choi ◽  
Yongchao Liu ◽  
Jagyeong Oh ◽  
Jiyeon Ha ◽  
Xuexiu Zheng ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Pcr Analysis ◽  
Splice Site Selection ◽  
Interaction Domain ◽  
Regulatory Functions ◽  
Auxiliary Factor

Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5′ splice-site selection, alternative 3′ splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.

Sign in / Sign up

Export Citation Format

Share Document