Don P. Bourque

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

Quantitative comparative analysis of complex two-dimensional electropherograms

Abstract A protocol is described to detect and assess differences between complex electrophoretic patterns. A semiautomated method is used to collect accurate absolute mobility data from many two-dimensional electropherograms and a computer algorithm has been developed which normalizes and averages these data. The program generates refined numerical maps consisting of the mean electrophoretic mobilities and corresponding confidence limits for each component protein represented in the original two-dimensional electrophoretic pattern. Tests of statistical significance of apparent differences between averaged numerical maps are carried out to evaluate electrophoretic polymorphisms between the ribosomal proteins of two different plant species. Furthermore, using a nonlinear function relating log molecular weight to mobility, precise molecular weight estimates are obtained from measurements of electrophoretic mobilities of proteins in the presence of sodium dodecyl sulfate. Several examples are presented which demonstrate application of these semiautomated analyses to quantitative comparison and interpretation of two dimensional gel electropherograms.

Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis

A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed.

Crystalline Ribulose 1,5-Bisphosphate Carboxylase-Oxygenase from Spinach

Spinach fraction I protein (ribulose 1,5-bisphosphate carboxylase-oxy genase, E.C. 4.1.1.39) was crystallized on both an analytical and a preparative scale by vapor diffusion with polyethylene glycol (molecular weight, 6000) used as the precipitant. The identity of the crystalline material with fraction I protein was shown by gel electrophoresis in the presence of sodium dodecyl sulfate and immunological properties. The carboxylase and oxygenase activities copurify during crystallization, and the crystalline enzyme lacks copper and iron.

High specific activity ribulose 1,5-bisphosphate carboxylase-oxygenase from Nicotiana tabacum

Ribulose 1,5-bisphosphate carboxylase (EC.4.1.1.39) has been obtained from Nicotiana tabacum leaf homogenates with specific activites from 0.5 to 0.8 µmol CO2 fixed (mg protein min)-1. These activities are reconciled with much lower, previously reported activities. The results suggest that if the tobacco enzyme is assayed under optimum conditions there is little difference in the intrinsic specific activities of tobacco and spinach ribulose 1,5-bisphosphate carboxylase. Several factors affecting activity measurements were examined.

A tobacco mosaic virus mutant with non-functional coat protein and its revertant: relationship to the virus assembly process.

A nitrous acid-induced coat protein-defective mutant (PM6) derived from the wild-type strain (U1) of tobacco mosaic virus (TMV) and its coat protein-functional revertant (PM6R) have been isolated. PM6 is a virus assembly mutant in which the coat protein cannot encapsidate viral RNA. The coat protein of PM6 forms unusual wheel-like structures consisting of aggregated disks with a helical conformation. PM6 protein has two amino acid substitutions when compared to U1-TMV, an Ala to Thr exchange at position 105 and an Asp to Gly exchange, probably at position 88. PM6R has the Ala to Thr exchange found in PM6 at position 105, but the Asp to Gly exchange observed in PM6 has apparently reverted to the native sequence found in U1-TMV. A high frequency of irregular protein rods is observed in electron micrographs of PM6R, suggesting that the conformation of PM6R coat protein, although functional, is altered from that of the wild-type strain.

An ethidium-acrylamide affinity medium for recovery of nucleic acids from free solution and from polyacrylamide and Agarose gels.

Abstract The simple preparation of an ethidium-bromide-based nucleic acid affinity medium is described. The medium is composed of an acrylamide matrix to which ethidium bromide is attached. Its use in preparative purification and fractionation of nucleic acids in solution and in electrophoretic elution of nucleic acids from gels is reported. Nucleic acids can be eluted from this medium with a buffered salt solution and concentrated by ethanol precipitation without persistent contamination with undesirable impurities.

Crested Saguaros: What is the Rhyme or Reason?

Of imposing size and appearance, the saguaro cactus ( Carnegiea gigantea ) is an emblem of the Sonoran Desert, home of the Reporter’s editorial office. This is the honored species of the Saguaro National Park, Tucson, Arizona, where stately plants are abundant in their natural habitat. Cristate specimens of saguaro and other cactus species are highly prized by collectors. Consequently they are now relatively abundant in urban Sonoran settings. This issue’s cover photograph of a crested saguaro was chosen to stimulate the curiosity of plant molecular biologists. A research opportunity exists to solve the question: What is the molecular basis of saguaro cristation? This subject could be considered a research opportunity, since the etiology of cristation is unknown. Because the life cycle of saguaro is orders of magnitude longer than that of Arabidopsis thaliana , a commitment to this research would require long term planning! There are few publications addressing the crested saguaro condition. Over three decades ago, Snyder and Weber (1966) summarized what was known about causative factors of cristation in saguaro and other Cactaceae species. Boke and Ross (1978) published an anatomical study of cristation in a related cactus species. One must conclude from these studies, and the lack of subsequent elucidation, that causes of this phenomenon are still obscure. Here is some information about cristation obtained from the cited references: • Cristation is a fasciation in which the top of a plant is malformed due to unusual development of the apical meristem. A saguaro plant becomes crested when its tip develops laterally from an elongate meristem rather than a single point. • Fasciation is common among vascular plants, being found in over 100 plant families and in more than 50 genera of cacti. (See photographs in cited references).

Mode of inheritance and evidence for cistron heterogeneity of chloroplast 16S ribosomal RNA genes in Nicotiana

Oligonucleotide maps (fingerprints) of T1 RNase digests of 125I-labeled 16 S chloroplast rRNA of Nicotiana tabacum and N. gossei revealed the presence of T1 oligonucleotide fragment 100 in the 16 S rRNA of N. gossei while N. tabacum 16 S rRNA had a unique T1 oligonucleotide (fragment 101) as well as some fragment 100. From the positions in the fingerprints and from fingerprints of secondary enzymatic digestion of the fragments, we conclude that fragments 100 and 101 are similar in sequence and size, but fragment 100 probably contains an extra uracil residue. This difference is shown to be maternally inherited, thus confirming the location of 16 S chloroplast rRNA genes on chloroplast DNA and ruling out the possibility of genetically active chloroplast rRNA genes in the nucleus. The presence of both fragments 100 and 101 in N. tabacum may indicate sequence heterogeneity between the two cistrons for 16 S chloroplast rRNA. These results demonstrate the feasibility of determining the inheritance of organelle genes by genetic analysis of their primary transcripts.

Improved preparative methods for isolation and purification of tobacco chloroplast ribosomes, ribosomal proteins, and rRNA

Publisher Summary This chapter describes the procedures for the isolation of chloroplast ribosomes on a preparative scale by zonal sucrose gradient centrifugation from the whole leaf extracts. Methods for extracting ribosomal proteins and ribosomal RNA (rRNA) from the purified ribosomes are also described. The protein-synthesizing machinery of chloroplasts is a class of ribosomes that are unique to the chloroplast and of a 70 S type with many structural and functional similarities to bacterial ribosomes. The prokaryotic-like ribosomes are present in green leaves in equimolar amounts, a relative to the 80 S cytoplasmic ribosomes that share common features with other eukaryotic cytosolic ribosomes. Chloroplast ribosomes differ from the 80 S cytosolic ribosomes in their susceptibility to dissociation into subunits at different levels of magnesium ion. Chloroplast ribosome monosomes can be isolated at higher magnesium concentrations. The magnesium concentration required to control the equilibrium between the monosome and its dissociated subunits depends on the concentration of the major monovalent cation present in the solution.