Donald L. Robertson

Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.

Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.

SYNTHESIS OF LANTHANIDE--IRON LAVES PHASES AT HIGH PRESSURES AND TEMPERATURES.

High pressures and temperatures were used to synthesize the previously unknown compounds PrFe2, NdFe2 and YbFe2. These compounds have the cubic MgCu2-type structure with lattice parameters 7.467, 7.452 and 7.239 A respectively. The relative importance of 4f bonding and size effects in the formation of LFe2 compounds (L = lanthanide) is discussed.

Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis

The nucleotide sequence of the Bacillus anthracis lethal factor (LF) gene (lef) has been determined. LF is part of the tripartite protein exotoxin of B. anthracis along with protective antigen (PA) and edema factor (EF). The apparent ATG start codon, which is located immediately upstream from codons which specify the first 16 amino acids (aa) of the mature secreted LF, is preceded by an AAAGGAG sequence, which is its probable ribosome-binding site. This ATG codon begins a continuous 2427-bp open reading frame which encodes the 809-aa LF-precursor protein with an Mr of 93,798. The mature secreted protein (776 aa; Mr 90,237) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. The codon usage of the LF gene reflects its high (70%) A + T content. The N-terminus of LF (first 300 aa) shared extensive homology with the N-terminus of the anthrax EF protein. Since LF and EF each bind PA at the same site, these homologous regions probably represent their common PA-binding domains.

Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis

We have cloned and expressed in Escherichia coli the lethal factor (LF) gene of Bacillus anthracis. At least two of the six LF recombinant plasmids produce full-length LF protein. Transcription of the LF gene in E. coli appears to be under the control of its own B. anthracis promoter. Recombinant LF protein produced in E. coli remains intracellular and is not secreted. However, this LF protein is biochemically active and displays the same lethal effects as LF secreted by B. anthracis in the mouse macrophage assay. The LF gene, like that of the protective antigen gene, is present on the large B. anthracis toxin plasmid pXO1.

Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase.

The nucleotide sequence of the Bacillus anthracis edema factor (EF) gene (cya), which encodes a calmodulin-dependent adenylate cyclase, has been determined. EF is part of the tripartite protein exotoxin of B. anthracis. An ATG start codon, immediately upstream from codons which specify the first 15 amino acids (aa) of EF, was preceded by an AAAGGAGGT sequence which is its probable ribosome-binding site. Starting at this ATG codon, there was a continuous 2400-bp open reading frame which encodes the 800-aa EF-precursor protein with a Mr of 92,464. The mature, secreted protein (767 aa; Mr 88,808) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. A consensus amino acid sequence (Gly-X-X-X-X-Gly-Lys-Ser,X = any aa), which was part of the presumed ATP binding site for EF, was also present. The codon usage of the EF gene reflected the high A + T (71%) base composition for its DNA. B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase.

Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2

Virulent strains of Bacillus anthracis contain two large plasmids. pXO1 encodes the three component protein exotoxin and pXO2 is necessary for synthesis of the poly-D-glutamic acid capsule. A procedure for the isolation of these plasmids which yields high quantities of pure DNA is described. Restriction endonuclease analysis of these plasmids shows that they are not related. pXO1 is 174 kilobase pairs and pXO2 is 95 kilobase pairs. From their bouyant densities and melting temperatures we also determined their GC contents. pXO1 contains 31.1% GC base pairs and pXO2 is 31.4% GC. Both of these values are close to the GC content of B. anthracis genomic DNA which is 32.2%.

The effect of high pressure on the formation of LRu2 and LOs2 (LLanthanide) compounds

Abstract The compounds SmRu 2 , NdRu 2 and LaOs 2 have the cubic MgCu2-type structure when prepared at atmospheric pressure. By application of pressures up to 88 kbar, these compounds were prepared with the hexagonal MgZn 2 -type structure. The hexagonal lattice parameters ( a , c ) are (5.298, 8.939), (5.323, 9.004), and (5.42, 9.00), respectively. The formation of these hexagonal polymorphs is attributed to the effect of pressure on the relative atomic size of the lanthanide vs. that of Ru or Os. The amount of 4 f bonding in these compounds has no influence on the type of polymorph formed.

HIGH-PRESSURE AND HIGH-TEMPERATURE SYNTHESIS OF LaCo2

Abstract High pressures and high temperatures were used to synthesize previously unknown LaCo 2 . This compound has the cubic MgCu 2 -type structure with a lattice parameter of 7.449 A. Attempts to prepare EuCo 2 and LaCo 3 were not successful.

Relationships between the calmodulin-dependent adenylate cyclases produced by Bacillus anthracis and Bordetella pertussis.

The nucleotide sequences for the calmodulin-dependent adenylate cyclases produced by Bordetella pertussis and Bacillus anthracis have recently been determined. The GC% for the B. pertussis and B. anthracis cyclase genes are about 65% and 29%, respectively. Despite this difference in nucleotide composition, these cyclases possess three highly conserved amino acid domains and share some nucleotide sequence homology. One of these conserved domains appears to be involved in ATP binding and is related to the consensus amino acid sequences present in many eukaryotic and prokaryotic ATP and GTP binding proteins. The possible relationship between these cyclases and eukaryotic calmodulin-dependent adenylate cyclases is discussed.

Complete cloning of the chloroplast genome of safflower in lambda EMBL3 and mapping of 23S and 16S rRNA genes.

SummaryA recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of λ EMBL3. Seventeen λ recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.