Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Sequence and expression of a human type II mesothelial keratin

Using mRNA from cultured human mesothelial cells, we constructed bacterial plasmids and lambda phage vectors that contained cDNA sequences specific for the keratins expressed in these cells. A cloned cDNA encoding keratin K7 (55 kD) was identified by positive hybrid selection. Southern Blot analysis indicated that this sequence is represented only once in the human genome, and Northern Blot analysis demonstrated that the gene encoding K7 is expressed in abundance in cultured bronchial and mesothelial cells, but only weakly in cultured epidermal cells and not at all in liver, colon, or exocervical tissue. The predicted amino acid sequence of this keratin has revealed a striking difference between this keratin and the type II keratins expressed in epidermal cells: whereas all of the epidermal type II keratins thus far sequenced have long nonhelical termini rich in glycine and serine, this mesothelial type II keratin has amino and carboxy terminal regions that are unusually short and lack the inexact repeats of glycine and serine residues.     

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).     

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.     

Radioprotective properties of detoxified lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).     

Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti

The distribution and biosynthesis of ecdysone and 20-hydroxyecdysone (20-OH-ecdysone) was followed in sugar- and blood-fed female Aedes aegypti. In both sugar- and early blood-fed animals most of the ecdysteroid determined by radioimmunoassay was found outside the ovary. Twenty-four to 40 h after blood feeding, however, ecdysteroid was distributed between ovary and carcass in the ratio of 1:1.5. Ecdysteroid titer reached a plateau between 18 to 40 h after the blood meal and decreased thereafter. Analysis of the ecdysteroid titer using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) revealed that both 20-OH-ecdysone and ecdysone were synthesized after the blood meal. The ratio of 20-OH-ecdysone to ecdysone remained essentially constant and fluctuated in parallel throughout egg development. Chromatography of the early ecdysteroid peak (8 h after feeding) using TLC and HPLC indicated that although it cross-reacted with ecdysteroid antibodies, it did not have the same elution times as ecdysone and 20-OH-ecdysone and is, therefore, probably a precursor of these ecdysteroids. Injections of egg development neurosecretory hormone (EDNH) preparation purified to near homogeneity, into ligated abdomens, induced ecdysteroid synthesis only if the abdomens were first treated with methoprene (12.5 pg). Methoprene at this concentration did not stimulate ecdysteroid synthesis in these abdomens. When blood-fed females were treated with [4-¹⁴C] cholesterol and analyzed using TLC and HPLC procedures, both [¹⁴C]labeled ecdysone and [¹⁴C]labeled 20-OH-ecdysone were synthesized in the ratio of 1:1.5. This report is the first to show that both ecdysone and 20-OH-ecdysone are synthesized in vivo in female A. aegypti.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins

We present the cDNA and amino acid sequences of a cytoskeletal keratin from human epidermis (Mr = 56K) that belongs to one of the two classes of keratins (Type I and Type II) present in all vertebrates. In these two types of keratins the central approximately 300 residue long regions share approximately 30% homology both with one another and with the sequences of other IF proteins. Within this region, all IF proteins are predicted to contain four helical domains demarcated from one another by three regions of beta-turns. The amino and carboxy termini of the Type II keratin are very different from those of microfibrillar keratins and other nonkeratin IF proteins. However, they contain unusual glycine-rich tandem repeats similar to the amino terminus of the Type I keratin. Thus the size heterogeneity among keratins appears to be a result of differences in the length of the terminal ends rather than the structurally conserved central region.