Search results for "cDNA"

Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6: an improved method to determine 5′- and 3′-cDNA ends

1999

Abstract Rapid cloning of 5′- and 3′-cDNA ends polymerase chain reaction (5′-/3′-RACE-PCR) is useful to determine unknown 5′- and 3′-cDNA termini. Even if the method can yield complete cDNA sequences within a couple of days, the RACE procedure bears some characteristic traps and often results in amplification of unspecific PCR-products. Here we used improved 5′- and 3′-RACE-PCR protocols to obtain the complete cDNA sequence of the G-protein-coupled receptor kinase 6 (GRK6) from a rat brain cDNA library. The use of an anchored oligo-(dT) 16 -V-primer in the cDNA synthesis, the addition of single-sided PCR steps prior to the RACE-PCRs and the optimization of the dA-tailing reaction conditions…

A frame shift mutation in a hot spot region of the nuclear autoantigen La (SS-B).

1996

A hot spot region was identified in the exon 7 of the nuclear autoantigen La (SS-B). Two La cDNAs were identified which contained a frame shift mutation in the hot spot region. One La cDNA was isolated from a cDNA library made from peripheral blood lymphocytes of an autoimmune patient with primary Sjogren's Syndrome, the other La cDNA was isolated from a human liver cDNA library. The patient's La cDNA had a deletion and the liver La cDNA had an insert of an (A)-residue at the same position. Inserts of 4, 16 and 24 more or less homogeneous (A)-residues were found at the same site in the three La retropseudogenes. The hot spot region located in one of the major autoepitope regions of the La a…

Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of it…

2000

A major 43kDa protein from the protective tube of Riftiapachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100–110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that se…

2020

ObjectiveThe HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin.DesignWe explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA.ResultsWe show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the…

Noncanonical RNAs from transcripts of the Drosophila muscleblind gene.

2006

It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. Here we describe new cDNAs from the Drosophila melanogaster muscleblind (mbl ) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2#) or having exons with a different order compared to the corresponding genomic DNA (mblE2E3#E2#; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomp…

Divergently Transcribed Overlapping Genes Expressed in Liver and Kidney and Located in the 11p15.5 Imprinted Domain

1998

Human chromosomal band 11p15.5 has been shown to contain genes involved in the development of several pediatric and adult tumors and in Beckwith-Wiedemann syndrome (BWS). Overlapping P1 artificial chromosome clones from this region have been used as templates for genomic sequencing in an effort to identify candidate genes for these disorders. PowerBLAST identified several matches with expressed sequence tags (ESTs) from fetal brain and liver cDNA libraries. Northern blot analysis indicated that two of the genes identified by these ESTs encode transcripts of 1-1.5 kb with predominant expression in fetal and adult liver and kidney. With RT-PCR and RACE, full-length transcripts were isolated f…

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodi…

1993

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various s…

De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

2013

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and L…

Complete coding nucleotide sequence of cDNA for the class II RT1.B beta I chain of the Lewis rat.

1991

We have established the first full length cDNA clone for the beta light chain of the MHC class II alpha, beta heterodimer (isotype RT1.B) of the rat. Clone pLR beta 118 was obtained from a self-primed lambda gt10 cDNA library of IFN-tau treated bone marrow-derived macrophages of the Lewis rat. Subcloning of pLR beta 118 into a transcription vector with subsequent in vitro transcription and translation using the reticulocyte lysate system in the presence of microsomes followed by immunoprecipitation with mAb OX6 and two-dimensional gel electrophoresis revealed the intact RT1.B beta I-chain.

Expression profiling of human fetal growth plate cartilage by EST sequencing.

2005

The differentiation of mesenchymal stem cells into hypertrophic chondrocytes is an integral and multistep process important in pattern formation, endochondral ossification, and postnatal growth of the skeleton. In recent years, novel genes involved in these processes have been identified, but still only little is known about the large-scale gene expression profile during skeletal development. We initiated an expressed sequence tag (EST) project aiming at the identification of genes and pathways involved in this complex process. Candidate genes are expected to be of value for diagnosis and treatment of monogenic and multigenic heritable disorders of the skeleton. Here, we describe the sequen…