The isolation and characterization of the soluble and membrane-bound porcine cytochrome b5 cDNAs.

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.

The isolation and characterization of the human cytochrome b5 gene.

From a series of lambda and cosmid libraries, we isolated DNA sequences corresponding to the complete coding region for the human cytochrome b5 (CYB5) gene. The overall gene organization was the same as the bovine and rabbit CYB5 genes. One cosmid clone containing exon I plus the 5' flanking region was extensively characterized. From this clone we obtained 2000 bp of 5' flanking sequence, containing several distinctive GC rich regions, potential trans-acting factor binding sites and a 74 bp direct repeat. A series of deletion constructs were made in the pGL2 luciferase vector and then used to transfect HepG2 and K562 cells. The data obtained suggest the presence of two promotors and one silencer region in the analyzed 5' sequence.

A splicing mutation in the cytochrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism.

We have analyzed reticulocyte and leukocyte mRNAs isolated from a patient with congenital methemoglobinemia and pseudohermaphrodism. The cytochrome b5 cDNA sequences were amplified using specific oligonucleotide primers and the polymerase chain reaction (PCR). DNA sequencing indicated that there was a 16-bp deletion in the cDNA leading to a new, in-frame stop signal and resulting in a truncated protein of 45 amino acids. Genomic DNA was analyzed, and the molecular lesion was shown to be an AG-->GG alteration in the 3' splicing junction of intron 1. The splice site alteration leads to the usage of the nearest AG as an alternative splice site, resulting in a 16-bp deletion in the mRNA. All of the studies on reticulocyte mRNA and genomic DNA indicated that the patient was homozygous for the lesion.

Stopped-flow fluorescence studies of the interaction of a mutant form of cytochrome b5 with lipid vesicles.

Cytochrome b5 binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.

Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties.

The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.

The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively.

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.

The isolation and characterization of the bovine cytochrome b5 gene, and a transcribed pseudogene.

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.

Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5.

Cytochrome b5 is a liver integral membrane protein that has now been expressed in, and isolated from, Escherichia coli. The structure-function relationships of the 43 amino acid membrane-binding domain (nonpolar peptide) have been examined in both native and mutant forms of the protein; in the latter, tryptophan residues at positions 108 and 112 were replaced by leucine. The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding domain was examined while the domain was bound to lipid vesicles. Both the lipid-bound mutant domain and lipid-bound native domain showed an irreversible increase in fluorescence above 50 degrees C. When the whole cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-exchangeable ("loosely" bound), conformation to a final, virtually unexchangeable ("tightly" bound), conformation. It has been suggested previously that the protein exists in a "looped back" conformation and a "bilayer penetrating" conformation. Although the present studies are not designed to determine the absolute conformations of the loose and tight forms, the changes observed in steady-state and frequency-modulated fluorescence and the lack of change in depth of Trp 109 in the bilayer are consistent with a movement of the C-terminal segment from a looped back to a bilayer penetrating conformation as the tight form is generated.

Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b5.

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.

In situ hybridization study of obesity-associated alteration in growth hormone mRNA levels.

In order to investigate whether the impaired GH secretion associated with obesity is due to a pituitary disorder we studied GH mRNA levels by in situ hybridization in genetically obese and lean Zucker rats. The levels of GH mRNA were at least two fold lower in obese rats in comparison to that in lean controls as quantified by both the scanning of autoradiographs of tissue sections and Northern blot analysis. Quantification of somatotrophs revealed no significant difference in their number between lean and obese rat pituitaries. It is therefore likely that the attenuated GH mRNA levels in genetically obese Zucker rats are due to a decrease in GH transcripts per somatotroph rather than a result of a pituitary defect involving a preferential decrease in somatotroph population.

Identification and nucleotide sequence of the leukocyte and reticulocyte forms of rabbit cytochrome b5 mRNA.

RNA extracted from rabbit leukocytes and reticulocytes was reverse transcribed and used in the Polymerase Chain Reaction technique along with primers designed to amplify the coding sequence of rabbit cytochrome b5. The resultant amplified products were subcloned and analyzed. Sequencing confirmed that leukocyte and liver cDNAs are homologous and encode the membrane-bound form of the protein. In contrast, reticulocytes exhibit a highly similar, but different mRNA which encodes the smaller, soluble cytochrome b5. This is the first example of a cytochrome b5 sequence from a tissue other than liver, erythrocyte or reticulocyte.

Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain.

Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.

The human liver and reticulocyte cytochrome b5 mRNAs are products from a single gene.

Using a combination of standard cDNA library screening techniques and the Polymerase Chain Reaction (PCR) we have isolated and sequenced DNA fragments corresponding to the human reticulocyte cytochrome b5 mRNA. The reticulocyte specific sequence codes for amino acids 97 and 98 only, with a TAA stop codon. The reticulocyte specific 3'non-translated sequence has 15 new nucleotides then utilizes the liver mRNA sequence from amino acid 97 onward. This indicates that the reticulocyte specific exon has 24 base pairs (bp). In addition, we have isolated sequences that are derived from a transcribed cytochrome b5 pseudogene. This transcript contains multiple mutations which prevent the synthesis of any functional protein.

Developmental changes in levels of growth hormone mRNA in Zucker rats.

Levels of pituitary growth hormone (GH) messenger RNA (mRNA) were compared in groups of genetically obese (fa/fa) and lean (Fa/-) littermate male Zucker rats at four different ages, 3, 5, 9, and 11 weeks, in order to determine the earliest age at which a difference between the two groups could be detected. No difference was seen in three-week-old animals. Five weeks of age was the earliest time at which the level of GH mRNA was significantly decreased in the obese rats; this decrease was present at all subsequent ages. Mean serum growth hormone levels were lower in obese animals at all ages, but the differences were not statistically significant because of the large individual variation associated with the pulsatile nature of GH release. The earliest occurrence of differences in GH mRNA level is later than some of the obesity associated abnormalities present in adipose tissue. The earliest time of the GH mRNA differences can be associated with the time when decreased protein deposition is initially seen in the obese rats. Because of this association, decreased GH mRNA may enhance the development of obesity.

Obesity- and sex-related alterations in growth hormone messenger RNA levels.

The secretion of growth hormone (GH) is abnormal in genetically obese Zucker rats. Measurements of pulsatile GH release and circulating GH levels in lean (Fa/?) and obese (fa/fa) rats have shown that both are reduced in the latter. We have studied pituitary GH gene expression in order to understand the role of GH synthesis in this abnormality. Obese animals have lower pituitary GH mRNA levels than lean controls. Within each genotype a sex difference was observed with the female animals having lower GH mRNA levels than the males. It is unlikely that the GH abnormality is due to a generalized pituitary defect because prolactin mRNA levels were the same in all four groups of rats.

The characterization of three types of partially processed mRNA and two pseudogenes for human liver cytochrome b5.

We have isolated cDNA clones corresponding to partially processed human liver cytochrome b5 mRNAs. All the clones contained poly(A) sequences, and one clone had a shorter 3' non-translated sequence, indicating the use of an alternative poly(A) addition signal. In addition, all the clones contained the coding information for amino acids 87-134; however, there were two types of intron junction adjacent to the coding sequence. Detailed analysis of the Type I clones showed that the Type II intron sequence was contained within the Type I sequence, but approximately 1000 bp 5' of the Type I intron-exon junction showed alternative splicing within this intron. In addition, we have isolated two pseudogenes which lack introns, suggesting the retroviral insertion of human liver cytochrome b5 mRNA sequences into the human genome.