Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans

We have cloned two different beta-tubulin sequences from the filamentous fungus Aspergillus nidulans. Each was used in the construction of transforming plasmids that carry the pyr4 gene of Neurospora crassa. We used these plasmids to transform a pyrG-strain of Aspergillus to uridine prototrophy. Both plasmids were shown to integrate site specifically into the homologous chromosomal sequences. We then used transformant strains in genetic crosses to demonstrate that one of the cloned beta-tubulin sequences was the benA beta-tubulin gene, which codes for the beta 1-and beta 2-tubulins. The other cloned beta-tubulin sequence was shown to be the structural gene for beta 3-tubulin by gene disruption and to participate in conidial development. This is the first report of a gene disruption by site specific, integrative recombination in Aspergillus nidulans.     

Analysis of the genetic requirements for inducible multiple-antibiotic resistance associated with the mar locus in Escherichia coli.

A series of novel genetic constructs derived from the marRAB operon was used to determine the role of this gene cluster in salicylate-inducible multiple-antibiotic resistance in Escherichia coli. Our findings indicate that regulated antibiotic resistance associated with this locus requires only the products of marR and marA, without any neighboring genes.     

Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli.

The most frequently occurring kringle 4 domain of human apolipoprotein (a), Kringle 4-subtype 2 (K4(2)), was expressed as a fusion protein with the maltose binding protein in Escherichia coli using the "tac" promoter. Although the fusion protein was expressed without a signal sequence, 25% was secreted into the periplasmic space; the remainder was found associated with the soluble cytosolic fraction. The fusion protein was readily isolated from whole cell lysate by amylose agarose affinity chromatography. Although a factor Xa cleavage site was engineered into the fusion protein, it was found that release of the K4(2) protein was most conveniently achieved by proteolysis with subtilisin A. The cleavage product produced in this way was shown to be intact K4(2) with only the first three amino acid residues of the leading flanking peptide missing, as judged by N-terminal sequence analysis. K4(2) was isolated from the hydrolysate by FPLC on a Mono-Q column with a yield of 170 +/- 30 micrograms/g wet cells. The resulting protein was monomeric in phosphate-buffered saline as judged by size-exclusion chromatography and appeared to be folded as shown by spectroscopic and immunological assays. The recombinant K4(2) did not bind to either lysine- or proline-Sepharose, suggesting that the ligand binding activities of lipoprotein (a) may reside in the other kringle domains of apolipoprotein (a).     

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase from Zea mays: General properties and regulation by tryptophan

In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn⁺⁺ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn⁺⁺and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Investigations on the turnover of adrenocortical mitochondria

Summary The effects of chronic administration of ACTH (up to 36 consecutive days) on the mitochondria of the zona reticularis of the rat adrenal cortex were investigated by stereologic techniques. It was found that ACTH induces two phases of hypertrophy of mitochondria alternating with two proliferative stages, which are associated with a significant decrease in the average volume of the organelles. It is suggested that, as in the zona fasciculata, ACTH controls the processes of growth and division of mitochondria in the zona reticularis. The mechanism underlying this action of ACTH as well as the differences between the responses to ACTH of the mitochondrial population of the two adrenal zones are discussed in the light of evidence indicating that mitochondria contain a complete genetic apparatus largely independent of nuclear control.The authors wish to thank Miss A. Coi and Mr. G. Gottardo for their excellent technical assistance. This work was partly supported by a contract with the CNR (C.T. 73.00663.04)     

Relevance of the amino acid conversions L144R (Zaragoza) and L159P (Zavalla) in the apolipoprotein A-I binding site for haptoglobin

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.