Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b5.
Agar Gel; Amino Acid Sequence; Animals; Base Sequence; Blotting; Cell Membrane/metabolism; Cytochromes b5/*genetics; Cytosol/metabolism; DNA/genetics/isolation & purification; Electrophoresis; Exons; Leukocytes/metabolism; Liver/*metabolism; Messenger/genetics/isolation & purification/*metabolism; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction/methods; Rabbits; Reticulocytes/metabolism; RNA; Southern
Total RNA was extracted from a variety of rabbit tissues and reverse transcribed for use in the polymerase chain reaction technique. Using primers designed to amplify the membrane-bound liver cytochrome b5 cDNA, products of two sizes were observed. Both hybridized strongly to a radiolabelled liver cytochrome b5 probe. Sequencing confirmed that the two types of cDNA product encoded the membrane-bound and the soluble forms of b5. Messenger RNA corresponding to the soluble cytochrome was detected in the lung, gallbladder and the adrenal gland, as well as in reticulocytes and bone marrow. This was an unexpected finding since the protein has been isolated only from erythrocytes. In contrast, membrane-bound cytochrome b5 mRNA was detected in all tissues tested, suggesting that the corresponding protein is ubiquitous in tissue distribution.
Giordano S J; Steggles A W
Biochimica et biophysica acta
1993
1993-02
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/10.1016/0167-4781(93)90274-h" target="_blank" rel="noreferrer noopener">10.1016/0167-4781(93)90274-h</a>
The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively.
*Chromosomes; Animals; Blotting; Cell Line; Chromosome Mapping; Cricetinae; Cytochromes b5/*genetics; DNA/analysis; Genetic/genetics; Human; Humans; Hybrid Cells; In Situ Hybridization; Molecular Sequence Data; Pair 14; Pair 18; Pair 20; Polymerase Chain Reaction; Pseudogenes/*genetics; Southern; Translocation
Using very high stringency hybridization conditions for the Southern blot hybridization analysis of hamster-human cell hybrid DNA, we were able to map the human cytochrome b5 gene and two of its pseudogenes (psgb(5)1 and psgb(5)2) unambiguously to chromosomes 18, 14, and 20. These localizations were confirmed and extended to 18q23, 14q31-32.1, and 20p11.2 by using a combination of nonisotopic in situ hybridization of chromosomal spreads and the polymerase chain reaction analysis of DNA samples isolated from somatic cell hybrids retaining deletions or translocations of chromosome 18.
Giordano S J; Yoo M; Ward D C; Bhatt M; Overhauser J; Steggles A W
Human genetics
1993
1993-12
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/10.1007/bf00420948" target="_blank" rel="noreferrer noopener">10.1007/bf00420948</a>
The isolation and characterization of the bovine cytochrome b5 gene, and a transcribed pseudogene.
*Pseudogenes; Amino Acid Sequence; Animals; Base Sequence; Blotting; Cattle/*genetics; Cytochromes b5/*genetics; DNA/genetics/isolation & purification; Exons; Gene Expression; Genetic; Genomic Library; Humans; Introns; Messenger/biosynthesis/metabolism; Molecular Sequence Data; Nucleic Acid; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Rabbits; Restriction Mapping; Reticulocytes/metabolism; RNA; RNA Precursors/chemistry/metabolism; Sequence Homology; Southern; Transcription
This is the first isolation and characterization of a cytochrome b5(b5) gene. The bovine b5 gene is quite large, spanning about 28 kb and contains six exons. One of these exons appears to code for a reticulocyte-specific sequence similar to that described for human and rabbit b5. All of the splicing junctions conform to the GT-AG consensus rule. The 5' flanking sequence has no obvious TATA box, two CAAT boxes, and contains several G:C-rich SpI motifs indicative of a house-keeping gene. In reticulocyte mRNA we found evidence for a transcribed b5 pseudogene, but could not detect sequences coding for the soluble form of b5. We conclude that the soluble form of b5 is derived from the membrane-bound b5 by a post-translational mechanism.
Cristiano R J; Giordano S J; Steggles A W
Genomics
1993
1993-08
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/10.1006/geno.1993.1331" target="_blank" rel="noreferrer noopener">10.1006/geno.1993.1331</a>
The isolation and characterization of the soluble and membrane-bound porcine cytochrome b5 cDNAs.
Amino Acid Sequence; Animals; Base Sequence; Blotting; Complementary/blood/*chemistry/*isolation & purification; Cytochromes b5/blood/*chemistry/genetics/*isolation & purification; DNA; Male; Membrane Proteins/*chemistry/genetics/*isolation & purification; Molecular Sequence Data; Solubility; Southern; Swine; Testis/enzymology
In some male pigs, there is an increased production of the testicular 16 androstene steroids which end up being concentrated in fatty tissue. When the meat is cooked, a disagreeable odor/flavor is produced, a phenomenon known as "boar taint." All boars selected for food production are castrated even though only ca 10% of boars may be "tainted." This has a high economic cost because castrated pigs convert food into meat less efficiently, the meat is fatter, and there is an increased mortality due to the castration procedure. Recent data has implicated an increased level of cytochrome b5 in the testes with the increased synthesis of the 16-androstene steroids. As an initial step in analyzing this process, we used 5' and 3' RACE PCR procedures to isolate, clone and sequence the cDNAs for the membrane-bound and soluble forms of porcine cytochrome b5.
VanDerMark P K; Steggles A W
Biochemical and biophysical research communications
1997
1997-11
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/10.1006/bbrc.1997.7599" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1997.7599</a>
Genomic cloning, sequencing, and analysis of the hamster cholesterol 7 alpha-hydroxylase gene (CYP7).
Amino Acid; Amino Acid Sequence; Animals; Base Sequence; Blotting; Cholesterol 7-alpha-Hydroxylase/*genetics; Cloning; Cricetinae; DNA/genetics/isolation & purification; Genomic Library; Humans; Liver/enzymology; Mesocricetus/*genetics; Messenger/biosynthesis/metabolism; Molecular; Molecular Sequence Data; Northern; Poly A/biosynthesis/metabolism; Rats; Restriction Mapping; RNA; Sequence Homology; Southern
Cholesterol 7 alpha-hydroxylase is the rate limiting enzyme in bile acid biosynthesis and plays an important role in cholesterol homeostasis. The Golden Syrian hamster has been used as an animal model for the study of atherosclerosis and cholesterol gallstone disease. We have screened a lambda DASH II hamster liver genomic library using a rat cDNA as a hybridization probe. A 14-kb genomic clone has been isolated and characterized by restriction mapping and Southern blot hybridization. The clone contained the full-length gene encoding cholesterol 7 alpha-hydroxylase together with an upstream sequence of approximately 5 kb. DNA sequencing and analysis of about 11 kb of the gene revealed that the hamster CYP7 gene consists of six exons and five introns, which have the same structures and sizes as predicted in the rat and human CYP7 genes. The nucleotide and deduced amino acid sequences of the hamster cholesterol 7 alpha-hydroxylase have a high sequence identity of about 90% to the rat and 82% to the human sequences. Particularly, exons 2, 5, and 6 are highly conserved among these species, thus reflecting the presence of some domains that are crucial for the activity of this unique enzyme. The putative cholesterol-binding region, an aromatic amino acid region, and the P450 heme-binding region are completely conserved. Comparison of the 250-bp 5'-flanking sequence to the corresponding region in the rat and human genes revealed a high degree of homology ranging between 71% and 82%. Next to the canonical TATA and CCAAT boxes are many consensus sequences (LF-A1, LF-B1, TGT3) for liver-specific or -enriched transcription factors (HNF4, HNF1, and HNF5, respectively) and an imperfect direct repeat of thyroid hormone responsive element (TRE), which is located between TGT3 and LF-B1. These sequence motifs are completely conserved among the rat, human, and hamster CYP7 genes. Several modified sterol regulatory element (SRE)-like sequences are located in the upstream flanking region and in the first intron. This highly conserved proximal promoter may play important roles in the transcription activity and in the regulation of the CYP7 gene by physiological agents, such as bile acids and steroid/thyroid hormones. This is the first report describing the complete nucleotide sequence and confirming the structure of a CYP7 gene.(ABSTRACT TRUNCATED AT 400 WORDS)
Crestani M; Galli G; Chiang J Y
Archives of biochemistry and biophysics
1993
1993-11
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/10.1006/abbi.1993.1537" target="_blank" rel="noreferrer noopener">10.1006/abbi.1993.1537</a>