scholarly journals Molecular analysis of multiple mutator-derived alleles of the bronze locus of maize.

Genetics ◽  
1989 ◽  
Vol 122 (2) ◽  
pp. 439-445 ◽  
Author(s):  
W E Brown ◽  
D S Robertson ◽  
J L Bennetzen
Keyword(s):  
Molecular Level ◽  
Blot Hybridization ◽  
Insertion Site ◽  
Covalent Modification ◽  
Site Specificity ◽  
Coding Region ◽  
Restriction Sites ◽  
Mutant Induction ◽  
Bronze Locus ◽  
Molecular Nature

Abstract Very few mutations derived from Mutator maize lines have been studied at the molecular level. The variety of Mu elements that can induce mutations, the relative frequency of mutant induction by insertion of a given class of Mu elements or by a Mu-induced genomic rearrangement, a possible intragenic insertion site specificity, and the molecular nature of reversion events are all unknown in the Mutator system. To address these questions, we have isolated several partially or fully inactivated bronze alleles from Mutator maize lines and structurally characterized them by gel blot hybridization of genomic DNA. The mutations were induced in three parental Bronze alleles which differ by polymorphisms flanking the coding region. Each of the 14 inactivated bronze mutants characterized was found to contain an insert which cross-hybridized with the transposable element Mu1. Detailed maps of 11 of these alleles revealed a 1.4-kb insert with restriction sites characteristic of Mu1. These Mu1 insertions were found dispersed throughout both of the Bronze exons and in either orientation relative to Bronze transcription. Stable and somatically unstable (mutable) mutant alleles differed with respect to the covalent modification of restriction sites within the inserted Mu1 element. Several germinal revertants of one mutable bronze allele, bzMum4, were isolated. These all were associated with excision of the Mu1 element from the affected locus.

Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life

1994 ◽  
Vol 14 (12) ◽  
pp. 8219-8228
Author(s):  
P Belgrader ◽  
J Cheng ◽  
X Zhou ◽  
L S Stephenson ◽  
L E Maquat
Keyword(s):  
Triosephosphate Isomerase ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Coding Region ◽  
Protein Coding ◽  
Codon Recognition ◽  
Reverse Transcription Pcr ◽  
Nonsense Codon ◽  
Bona Fide ◽  
Nonsense Codons

Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.

scholarly journals cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene

1996 ◽  
Vol 318 (2) ◽  
pp. 689-699 ◽  
Author(s):  
Leonard DODE ◽  
Frank WUYTACK ◽  
Patrick F. J. KOOLS ◽  
Fouzia BABA-AISSA ◽  
Luc RAEYMAEKERS ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Blot Hybridization ◽  
Cdna Probe ◽  
Human Platelets ◽  
Gene Transcript ◽  
Northern Blot Hybridization ◽  
Dna Encoding ◽  
Coding Region ◽  
Cos Cells

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5´ region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3´-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5´-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177–6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

scholarly journals Insertion of the βGeo Promoter Trap into the Fem1c Gene of ROSA3 Mice

2004 ◽  
Vol 24 (9) ◽  
pp. 3794-3803 ◽  
Author(s):  
Cassandra L. Schlamp ◽  
Andrew T. Thliveris ◽  
Yan Li ◽  
Louis P. Kohl ◽  
Claudia Knop ◽  
...  
Keyword(s):  
Cell Death ◽  
Ganglion Cells ◽  
Neuronal Cell Death ◽  
Insertion Site ◽  
Neuronal Cell ◽  
Pyramidal Cells ◽  
Coding Region ◽  
Protein Levels ◽  
Molecular Events ◽  
Promoter Trap

ABSTRACT ROSA3 mice were developed by retroviral insertion of the βGeo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of βGeo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for βGeo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the βGeo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1cR3/R3 ) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1cR3/R3 mice may still be able to express a putative FEM1C protein. To this extent, Fem1cR3/R3 mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of βGeo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the βGeo reporter gene a potentially useful marker to study early molecular events in damaged neurons.

scholarly journals Hereditary Variation in Platelet Integrin α2β1 Density Is Associated With Two Silent Polymorphisms in the α2 Gene Coding Sequence

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 1939-1943 ◽  
Author(s):  
Thomas J. Kunicki ◽  
Marcie Kritzik ◽  
Douglas S. Annis ◽  
Diane J. Nugent
Keyword(s):  
Blood Platelets ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Type I ◽  
Receptor Density ◽  
Coding Region ◽  
Gene Frequencies ◽  
Coding Sequence ◽  
Hereditary Variation ◽  
Donor Population

Abstract The integrin α2β1 is a receptor for collagen that plays a fundamental role in the adhesion of blood platelets to the extracellular matrix. We previously reported that platelet α2β1 levels among randomly selected individuals can vary up to 10-fold and that this correlates with differences in adhesiveness to type-I or type-III collagens. We have now found two linked, allelic polymorphisms within the coding sequence of the α2 gene that correlate with receptor density, TTT/TTC at codon Phe224 and ACA/ACG at codon Thr246. By Southern blot hybridization of specific antisense DNA probes to segments of genomic DNA that encompass each coding region, we have determined the gene frequencies of each allele in a random donor population (n = 65) to be 0.585 (TTC ... ACG) and 0.415 (TTT ... ACA). There is a statistically significant correlation between the alleles TTT ... ACA (codons 224…246) and high receptor density (n = 30; P < .002), whereas the complimentary alleles TTC ... ACG are associated with low receptor density. Heterozygous individuals express intermediate levels of this receptor, and familial studies confirm that these allelic polymorphisms are inherited characteristics. These findings prove that the level of platelet α2β1 is an inherited trait. The molecular basis for receptor density remains to be determined, but our findings establish that these silent alleles within the coding sequence of the α2 gene are linked to the genetic basis for variation in receptor density.

scholarly journals Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 70-76 ◽  
Author(s):  
RW Kuijpers ◽  
S Simsek ◽  
NM Faber ◽  
R Goldschmeding ◽  
RK van Wermerkerken ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Single Point ◽  
Blot Hybridization ◽  
Platelet Glycoprotein ◽  
Single Point Mutation ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Dot Blot ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

Abstract Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called “Mo.” By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.

Cloning and Characterization of Chicken CRBP2 Gene and Comparison with other Vertebrates

2011 ◽  
Vol 343-344 ◽  
pp. 678-682
Author(s):  
Li Hua Xiao ◽  
Fan Li Kong ◽  
Hua Dong Yin ◽  
Xiao Ling Zhao ◽  
Qing Zhu
Keyword(s):  
Vitamin A ◽  
Egg Production ◽  
Binding Protein ◽  
Retinol Binding Protein ◽  
Production Traits ◽  
Coding Region ◽  
Small Intestinal ◽  
Small Intestinal Villus ◽  
Molecular Nature

Cellular retinol-binding protein 2 (CRBP2), a vitamin A binding protein expressed specifically in small intestinal villus absorptive cells, plays a pivotal role in the intestinal vitamin A absorption, transport, and metabolism pathways. In this study, we cloned the entire coding region of chicken CRBP2 gene. The amplified fragment contains entire coding region sequence with 408 nucleotides, which putatively codes 135 AA. By comparing nine vertebrates, the homology of nucleotide sequences is from 52.3% to 99.8%, while the similarity of AA sequence ranged from 72.4% to 99.3%. Results showed that the CRBP2 gene was conservative among different animal species. This work constructed the basis for further research on the molecular nature and genetic markers of CRBP2 for improving egg production traits in chicken.

Differentiation of Schistosoma mansoni from S. rodhaini using cloned DNA probes

Parasitology ◽  
1989 ◽  
Vol 98 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Tina K. Walker ◽  
A. J. G. Simpson ◽  
D. Rollinson
Keyword(s):  
Schistosoma Mansoni ◽  
Blot Hybridization ◽  
Repeated Sequence ◽  
Transcription Unit ◽  
Hybridization Analysis ◽  
Rrna Gene ◽  
Dot Blot ◽  
Restriction Sites ◽  
Genes Encoding ◽  
Female Specific

SummaryThe ribosomal RNA (rRNA) gene units of Schistosoma mansoni and S. rodhaini, of the lateral spined egg group, have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit between the two species is demonstrated to take the form of loss or gain of restriction sites within the non-transcribed and transcribed spacers. This variation has been demonstrated to enable the differentiation of S. mansoni from S. rodhaini by Southern hybridization analysis. In addition, a DNA clone representing a female specific, tandemly repeated sequence, is demonstrated to enable differentiation of S. mansoni and S. rodhaini using dot blot hybridization analysis.

scholarly journals Using of Data Base to Determine the Restriction Sites and Drawing Restriction Map for Lipase Gene from Bacillus stearothermophilus

2011 ◽  
Vol 5 (1) ◽  
pp. 22-30
Author(s):  
Hameed M. Jasim
Keyword(s):  
Data Base ◽  
Bacillus Stearothermophilus ◽  
Recombinant Dna ◽  
Restriction Enzymes ◽  
Complete Sequence ◽  
Restriction Map ◽  
Coding Region ◽  
Lipase Gene ◽  
Basic Language ◽  
Restriction Sites

pecific data base was used for restriction enzymes (rebase) and related proteins, to design executive program in quick basic language to determine the restriction sites and drawing restriction map for the complete sequence of lipase gene from Bacillus stearothermophilus. This program which was of a great benefit in prediction of the restriction patterns for the analysis of recombinant DNA molecules was used to determine the restriction sites for 16 enzyme among 46 type of different restriction enzymes having recognition sequences included in lipase gene which they are SmaI, XhoI, SalI, DdeI, ClaI, EagI, BspHI, AccI, EcoRV, HaeII, BanI, BclI, CfrI , SstI, KspI and NaeI. Results showed that it could be determine accurately the restriction sites and fragments sizes resulted in case of the treatment of lipase gene with these restriction endonucleases. It was also enabled to draw restriction map specific for these enzymes and determine coding region for lipase gene sized of 1254 bp included in the complete sequence for the cDNA of the gene sized of 1725 bp.

scholarly journals Distinguishing the forces controlling genetic variation at the Xdh locus in Drosophila pseudoobscura.

Genetics ◽  
1989 ◽  
Vol 123 (2) ◽  
pp. 359-369 ◽  
Author(s):  
M A Riley ◽  
M E Hallas ◽  
R C Lewontin
Keyword(s):  
Genetic Variation ◽  
Linkage Disequilibrium ◽  
Base Pair ◽  
Natural Populations ◽  
Xanthine Dehydrogenase ◽  
Drosophila Pseudoobscura ◽  
Common Haplotype ◽  
Coding Region ◽  
Data Set ◽  
Restriction Sites

Abstract Fifty-eight isochromosomal lines sampled from two natural populations of Drosophila pseudoobscura in California and one from Bogota, Colombia, were examined using four-cutter restriction mapping. A 4.6-kb region of the xanthine dehydrogenase locus was probed and 66 of 135 restriction sites scored were polymorphic. This predicts that on average every 12th bp would be polymorphic in this region for the genes surveyed if polymorphism occurred randomly along the coding region. In addition, there were 12 insertion/deletion polymorphisms. Forty-nine distinct haplotypes were recognized in the 58 lines examined. The most common haplotype obtained a frequency of only 5%. Measures of base pair heterozygosity (0.0097) and linkage disequilibrium lead to a predicted population size in the range of 1.2-2.4 X 10(6) for the species. High levels of recombination (including gene conversion) can be inferred from the presence of all four gametic types in the data set.

scholarly journals Complete Nucleotide Sequence of pSK41: Evolution of Staphylococcal Conjugative Multiresistance Plasmids

1998 ◽  
Vol 180 (17) ◽  
pp. 4350-4359 ◽  
Author(s):  
Tracey Berg ◽  
Neville Firth ◽  
Sumalee Apisiridej ◽  
Anusha Hettiaratchi ◽  
Amornrut Leelaporn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Nucleotide Sequence ◽  
Transfer Functions ◽  
Insertion Site ◽  
Conjugative Plasmid ◽  
Site Specificity ◽  
Significant Group ◽  
Resistance Determinants ◽  
Transposon Insertion ◽  
Clinically Significant

ABSTRACT The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from Staphylococcus aureus, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn552-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS257-mediated cointegrative capture of other plasmids.

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