Genetic recombination of Xenopus laevis 5 S DNA in bacteria.

The behavior in genetic recombination of Xenopus laevis 5 S DNA has been examined, with particular emphasis on the role of 15-base-pair tandem repeats in the A + T-rich spacer. Fragments of 5 S DNA were introduced into Escherichia coli cells as inserts in the recombination vectors, lambda rva and lambda rvb. Intermolecular recombinants were selected in which, because of properties of the phage vectors, the crossover event must have occurred within the 5 S DNA inserts. Inserts from individual recombinants have been characterized in detail. The effects of varying the number (n) of 15-base-pair repeats and the recombination capabilities of the phage and host have been investigated. In these crosses, unequal crossovers can occur, yielding inserts different in size from the parental inserts. When the number of 15-mers is large (n = 12 or 20), most of the unequal crossovers have occurred within the 15-mers, resulting in an altered n value, although other homologies within the 5 S DNA sequence can also support unequal events. Increasing n in the parental inserts modestly increases the overall frequency of recombination and the percentage of altered inserts. We conclude that, in a bacterial setting, the 15-base-pair repeats stimulate recombination only slightly by allowing alternative registers for heteroduplex formation. The degree of stimulation observed is less than predicted by one simple model.

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

A protein induced by NaCl in suspension cultures ofNicotiana tabacum accumulates in whole plant roots.

We have characterized a 26 000 dalton (26 000 D) protein which accumulates inNicotiana tabacum cuspension cells grown in media containing 10-25 g/l NaCl (7, 11, 17). Antibody was prepared against this protein and used to examine protein accumulation in both suspension cells and whole plants. Western blot analysis revealed that the 26 000 D protein also accumulates in suspension cells grown in the absence of NaCl as they approach stationary phase but the accumulation never reaches the level seen in the salt adapted cells. This protein also accumulates after treatment with other agents which lower the water potential, such as PEG and KCl, but no increase is seen after nonosmotic stresses such as heat shock and growth in cadmium chloride. The 26 000 D protein is found not only in whole tobacco plants but also in other members of the Solanaceae that were tested, as well as in alfalfa and green beans. The accumulation of the protein seems to be tissue specific as there is considerably more accumulation in roots than in stems or leaves of greenhouse grown plants. We have been unable to correlate accumlation of the 26 000 D protein with salt in wild tomato species but have demonstrated an increase in the accumulation of this protein with salt stress in hydroponically grown tomato plants. These results lead to speculation as to the role of this protein in responding to lowered water potential in the whole plant.

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein.

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH2-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5' signal sequence, an open reading frame of 718 nucleotides, a 3' AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.

Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.