Functional consequences of the excision of an omega loop, residues 40-55, from mitochondrial cytochrome c

A novel technique for protein semisynthesis, enzymic activation, has been used to create a mitochondrial cytochrome c analogue in which the conventional bottom loop has been deleted. The resulting structure resembles that of cytochrome c555 from a primitive photosynthetic sulfur bacteria. Comparisons of this analogue with natural cytochromes show which of the functional differences between cytochromes c and c555 may be related directly to the incorporation of the loop. The structure is an example of an omega loop, recently defined as a discrete category of protein secondary structure. The analogue maintains the overall structure of the parent protein, but a significant change in redox potential has been engineered. It provides support for the prediction that omega loops act as independent modules in folding, function, and evolution. The rapidity of the synthesis and the high yield of product show that this technique for protein engineering is both competitive with, and complementary to, genetic methods.     

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.     

Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

Summary A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the thylakoid-transfer domain of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.     

A search for trade-offs among life history traits inDrosophila melanogaster

Summary Are there underlying developmental and physiological properties of organisms that can be used to build a general theory of life history evolution? Much of the theoretical work on the evolution of life histories is based on the premise of negative developmental and genetic correlations among life history traits. If negative correlations do not exist as a general rule then no general theory taking them into account is possible. Negative genetic correlations among life history traits can come about by antagonistic pleiotropy. One cause of antagonistic pleiotropy is cost allocation trade-offs. Since cost allocation trade-offs are due to underlying physiological constraints they are expected to be common to closely related groups. A second form of antagonistic pleiotropy is specialization of genotypes to different niches. This type of antagonistic pleiotropy is expected to be specific to each population. We looked for trade-offs in life history traits of longevity and fecundity inDrosophila melanogaster. We used a half-sib mating design and raised the offspring at two temperatures, 19°C and 25°C. Correlations between longevity and fecundity showed some evidence of antagonistic pleiotropy at high temperature with no evidence of any trade-offs at low temperature. Correlations of early and late fecundity traits did show evidence of cost allocation trade-offs at both temperatures. Antagonistic pleiotropy was also found for cross-environmental correlations of fecundity traits. We conclude that, although life history trade-offs can not be generally assumed, they are frequently found among functionally related traits. Thus, we provide guidelines for the development of general theories of life history evolution.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.