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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The complete nucleotide sequence of human liver cytochrome b5 mRNA.

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.

The nucleotide sequence of rabbit liver cytochrome b5 mRNA.

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.

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes.

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups.

Upper genital tract abnormalities in the Syrian hamster as a result of in utero exposure to diethylstilbestrol. II. Scanning electron microscope analysis of endometrium cell surfaces.

Using scanning electron microscopy (SEM), we show that an in utero exposure to diethylstilbestrol (DES) will induce endometrial abnormalities postnatally. These changes are magnified when a subsequent postnatal DES treatment is given. More specifically, changes in cell surface morphology are associated with alterations in cell size and shape (from columnar to cuboid/squamous), and in microvilli and mucus secretion.