The complete nucleotide sequence of human liver cytochrome b5 mRNA.
Humans; Amino Acid Sequence; Liver/*metabolism; Base Sequence; Molecular Sequence Data; DNA/genetics; Cytochrome b Group/*genetics; Cytochromes b5; RNA; Cloning; Molecular; Messenger/*genetics
We have isolated and sequenced a cDNA clone corresponding to human liver cytochrome b5 mRNA. The 760 base pair (bp) sequence contains the complete coding and 3' non-translated regions plus 52 bp of 5' non-translated sequence. The derived amino acid sequence showed that the previous assignment of several amino acids was in error. In addition, the sequence of the previously unknown COOH hydrophobic region has been obtained.
Yoo M; Steggles A W
Biochemical and biophysical research communications
1988
1988-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/s0006-291x(88)80881-7" target="_blank" rel="noreferrer noopener">10.1016/s0006-291x(88)80881-7</a>
Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.
Female; Animals; Rats; Amino Acid Sequence; *Gene Expression Regulation; Kinetics; Base Sequence; Immunoblotting; Molecular Sequence Data; Steroid Hydroxylases/*genetics; Circadian Rhythm; DNA/genetics/isolation & purification; Restriction Mapping; Enzyme Induction; Liver/drug effects/*enzymology; Cell Fractionation; Cholesterol 7-alpha-Hydroxylase/biosynthesis/*genetics/immunology; Cholestyramine Resin/pharmacology; Cytochrome P-450 Enzyme System/genetics; Epitopes/analysis; Polyribosomes/metabolism/ultrastructure; Inbred Strains; RNA; Enzymologic; Sequence Homology; Cloning; Nucleic Acid; Messenger/*genetics; Centrifugation; Density Gradient; Molecular/methods
Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.
Li Y C; Wang D P; Chiang J Y
The Journal of biological chemistry
1990
1990-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
The nucleotide sequence of rabbit liver cytochrome b5 mRNA.
Animals; Amino Acid Sequence; Liver/*metabolism; Rabbits; Base Sequence; Molecular Sequence Data; Cytochrome b Group/*genetics; Cytochromes b5; Chromosome Deletion; Genes; RNA; Cloning; Molecular; Messenger/*genetics
Using a synthetic 26 base pair (bp) oligonucleotide, we have screened a rabbit liver cDNA library in lambda gt11 and isolated a 1000 bp DNA clone corresponding to cytochrome b5 mRNA. The clone contains the complete coding region and long 5' and 3' non-translated regions. The derived amino acid sequence (sequence; see text) agrees with published sequences, and confirms that the amino acids 62 and 104 are asparagine and aspartic acid, respectively.
Dariush N; Fisher C W; Steggles A W
Protein sequences & data analysis
1988
1905-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Isolation and characterization of cDNA clones for human, bovine and rabbit liver cytochrome b5 mRNA's.
Humans; Animals; Amino Acid Sequence; Rabbits; Base Sequence; Molecular Sequence Data; Liver/*enzymology; Cattle; Chickens; Cytochromes b5/blood/*genetics; DNA/*isolation & purification; Erythrocytes/enzymology; RNA; Sequence Homology; Cloning; Molecular; Nucleic Acid; Messenger/*genetics
Cristiano R J; Yoo M; Dariush N; Steggles A W
Progress in clinical and biological research
1989
1905-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
An effective and efficient method of transfecting primary human chondrocytes in suspension.
*Chondrocytes; *Osteoarthritis; *siRNA; *Transfection; Aggrecans/genetics; Articular/metabolism/pathology; Cartilage; Cells; Chondrocytes/cytology/*metabolism; Collagen Type II/genetics; Cultured; Humans; Messenger/*genetics; mRNA Cleavage and Polyadenylation Factors/antagonists & inhibitors/genetics; Nucleotidyltransferases/antagonists & inhibitors/genetics; Plasmids/*genetics; RNA; Small Interfering/*genetics; Transfection/*methods
Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.
Makki Mohammad Shahidul; Akhtar Nahid; Haqqi Tariq M
Analytical biochemistry
2017
2017-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ab.2017.03.009" target="_blank" rel="noreferrer noopener">10.1016/j.ab.2017.03.009</a>