ACTH treatment of bovine adrenocortical cells in primary culture causes increased accumulation of mRNAs encoding cytochromes P-450SCC, P-450(11)beta, P-450(17) alpha, P-450C21 and adrenodoxin as well as increased transcriptional activity of their respective genes. In this study we have shown that ACTH does not greatly affect the half-life of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin. However, in the case of P-450SCC mRNA, ACTH causes a five-fold increase in the half-life leading to a significant stabilization of P-450SCC mRNA. Thus it appears that the levels of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin are regulated by ACTH primarily at the transcriptional level, while that for P-450SCC is regulated at both the transcriptional and post-transcriptional levels.

The complete exonic and partial intronic sequence of the bovine CYP17 (P45017α) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when ~500 bp of the 5′-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450sec) and 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) in the bovine ovary. P-450sec is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450(17)alpha is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450scc, P-45011βP-45017αP-450c21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450scc, P-45017α and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85–145 days) showed constant levels of P-450scc and P-45011α, but increasing P-45017α content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Utilization of cDNA probes specific for various components of the bovine adrenocortical steroidogenic pathway have led to the conclusion that there are three levels of regulation of steroid hydroxylase gene expression. In each case it is postulated that specific classes of proteins bind to regulatory regions of these genes and modulate their transcription. Throughout adult life, cAMP-dependent regulation via SHIP protein(s) is the predominant mechanism by which optimal steroidogenic capacity is maintained. A second type of regulation is tissue-specific. One subclass of tissue-specific expression is the "all-or-none" type whereby steroid 21-hydroxylase and 11 beta-hydroxylase gene expression occur only in adrenal cortex and not in other steroidogenic tissues. A second subclass of tissue-specific expression is the "variable" type whereby 17 alpha-hydroxylase and cholesterol side chain cleavage (SCC) activity are both expressed in ovarian thecal cells but only SCC activity is expressed in corpus luteum. The third type of regulation is cAMP-independent and leads to fetal-imprinting (initial expression of steroid hydroxylase genes during fetal life).

Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues.

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species approximately equal to 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.

Kinetic and EPR studies show that the first step in the reaction of NO with ferric myoglobin, opossum hemoglobin, and microperoxidase is the reversible formation of the H-NO complex: H + NO in equilibrium H-NO (where H = Mb+, or Hb+ OP, or MP+). The NO-combination rates are markedly affected by the presence or absence of the distal histidine. The distal histidine significantly reduces the NO-combination rates, perhaps by interaction between the distal histidine and the ferric iron. Thus the beta-chains of Hb+ OP and metmyoglobin show similar combination rates. In the absence of a distal histidine, the NO-combination rates in the alpha-chains of Hb+ OP are much faster and similar to those observed for the five-coordinate heme in microperoxidase. The loss of a water molecule from the six-coordination site is assumed to be the rate-limiting step.

cDNA/RNA hybridization experiments of polysomal and nuclear poly(A)-rich RNA from early tadpole stages of Xenopus laevis revealed that part of the nuclear poly(A)-rich RNA sequences are not present within the polysomal polyadenylated RNA. For a more detailed analysis of these sequences we have cloned double-stranded cDNA derived from tadpole nuclear poly(A)-rich RNA in the PstI cleavage site of pBR 322. By colony screening with 32P-labelled cDNA from polysomal and nuclear poly(A)-rich RNA of the tadpole stage we could identify and isolate some of the cloned sequences, which are present only within the nuclear RNA. However, hybridization with cDNA from polysomal poly(A)-rich RNA of the gastrula stage indicated that at least one of those sequences which are confined to the nucleus at tadpole stage may serve as mRNA at gastrula stage. We present evidence that nuclear and polysomal poly(A)-rich RNA molecules containing the same nucleotide sequence differ in size and that size reduction at the level of processing precedes and may enable cytoplasmic export. We conclude that, besides stage-specific regulation of transcription, post-transcriptional control mechanisms are also involved in gene expression during embryonic development.

1.1. Isolated opossum hemoglobin is found to be more susceptible to oxidation by several oxidizing agents than is human hemoglobin A.2.2. Opossum methemoglobin is found to be more susceptible to reduction by several reducing agents than is human hemoglobin A.3.3. These results are due to amino acid differences between the two hemoglobins, including the replacement of the distal histidine residue in the alpha chains of hemoglobin A by a glutamine residue in these subunits of opossum hemoglobin.4.4. Such differences also appear to be responsible for the ability of the opossum red cell NADH methemoglobin reductase to maintain opossum hemoglobin in the functional ferrous form.

Abstract The nitric oxide complex of hagfish hemoglobin exhibits electron spin resonance spectra centered around g = 2 with rhombic symmetry. The six coordinated spectrum is not influenced by protonation or presence of inositol hexaphosphate. Thus, the critical substitutions at E 7 (His → Gln) and E 11 (Yal → Ile) do not disrupt the proximal histidineiron bonds in the nitrosyl complex of this primitive hemoglobin, though the same type of substitution are known to cause destabilization of R quaternary structure in tetrameric mammalian nitrosylhemoglobins. This difference could be related to the difference in tension existing in the respective hemes of tetrameric and monomeric hemoglobins.

Phenylhydrazine-induced anaemia in goats and certain sheep carrying a βA-globin gene, leads to a switch from adult βA-globin mRNA to βC mRNA synthesis1. The βC globin chains are also transiently produced late in fetal life. The nucleotide sequence of selected fragments of cloned goat βC globin gene2 corresponds to the amino acid sequence determined some years ago3. A similar reactivation of globin genes has been reported for the baboon, Papio cynocephelus, where anaemic stress also leads to the production of fetal haemoglobin4. In the case of anaemic chicken, results of cDNA cloning and nucleotide sequencing experiments suggested that a novel type of α-globin gene, the ‘stress’ gene, is expressed5. The existence of such a gene was mainly supported by the fact that the cDNA nucleotide sequence did not agree with any known chicken α-globin chain amino acid sequence. In contrast, we now report results, including a reinvestigation of the αA-globin amino acid sequence, which reveal that in the anaemic chicken, the αA gene is expressed, and there is no evidence for a specific stress α-globin gene.

In the hemoglobin of the opossum, the alpha chains have different residues at positions E7 and E11 than do most other mammalian hemoglobins. In the opossum, the usual histidine at alpha E7 is replaced by glutamine, the valine at alpha E11 by isoleucine, and the hemoglobin is known to have a low oxygen affinity and a low Hill coefficient. Comparison of kinetic studies of opossum hemoglobin with normal human hemoglobin shows that alpha chains in Hb opossum, despite the lack of distal histidine, do not differ significantly in CO-combination rates in either the T or R states. These rates are much slower than the rates reported for Hb Zurich, the hemoglobin from Chironomus thumi thumi, or the monomeric components of glycera hemoglobin, all of which also have a different residue at E7. As compared with Hb A, the changes in ligand affinities in the T and R states are small and cannot account for the unusually high values of p50 for Hb opossum. The equilibrium and kinetic data indicate that the L = (T)/(R) is about 100 times higher for Hb opossum than for Hb A; CCO = KR/KT approximately equal to 0.014. The kinetic data on l'4 and l also indicate that the R leads to T equilibrium for Hb4(CO)4 and Hb4(CO)2 can be shifted in either direction by adding inositol hexaphosphate or by changing the pH.

The oxygen equilibrium properties of rat total hemoglobin show pH dependence. Thus oxygen affinity and cooperativity, which are significantly reduced at pH 6.0, show increase with increasing pH. Conformational studies using nitrosyl derivatives indicate that in rat nitrosyl hemoglobin the R to T equilibrium is shifted towards the T state in going from pH 7.0 to pH 6.0. Under similar conditions human hemoglobin A shows no significant changes in cooperativity or conformation. These results indicate that a destabilized oxy structure (R) exist in rat hemoglobins at pH 6.0. Alterations in the heme pockets and alpha 1 beta 2 contact points could lead to these structural characteristics. Crystallization of rat hemoglobins is dependent on an oxy-like structure since all liganded ferrous derivatives and methemoglobin form crystals in the pH range of 7.0-8.0. Deoxygenation of oxyhemoglobin crystals or addition of inositol hexakisphosphate to nitrosyl or methemoglobin derivatives result in solubilization of the crystals. Thus the quaternary and/or tertiary structural changes involved in the transition of a R to T state disrupt the crystal structure. The precise positioning of the complementary sites involved in the interaction of amino acids between rat hemoglobin tetramers seems to occur only when the molecules are in the quaternary or tertiary R conformation. This is in contrast to the polymerization of human deoxyhemoglobin S, where gel formation occurs only in the T state.