A new allele of the duplicated 27kD zein locus of maize generated by homologous recombination.

The 27kD zein storage protein locus in many inbred lines of maize consists of a tandem duplication of 12kb, with an expressed gene in each repeat, termed A and B. A single-copy allele with only the A gene can be generated from this duplication in particular stocks of the maize inbred line A188 by a mitotic process that includes a crossover at the 3' regions of the two genes (1). Here, we characterize a second single-copy allele with only the B gene, found in different stocks of A188. This allele arises from a homologous recombination at the highly conserved 5' regions of the two repeats, and cloning and sequencing of this allele define the crossover region. The A and B genes in the duplicated allele were previously shown to be expressed at different levels; this difference remains unchanged in either recombinant allele. Therefore, the crossover points of these two recombinant alleles define the borders of cis-acting sequences responsible for the differential expression of the two genes.     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

Region-Specific Cis- and Trans-Acting Factors Contribute to Genetic Variability in Meiotic Recombination in Maize

Understanding the genetic basis for variability in recombination rates is important for general genetic studies and plant-breeding efforts. Earlier studies had suggested increased recombination frequencies in particular F(2) populations derived from the maize inbred A188. A detailed phenotypic and molecular analysis was undertaken to extend these observations and dissect the responsible factors. A heritable increase in recombination in the sh1-bz1 interval was observed in these populations. A factor causing an approximate twofold increase mapped to the A188 Sh1-Bz1 region, behaved as a dominant, cis-acting factor, affected recombination equally in male and female sporogenesis and did not reduce the wellstudied complete interference in the adjacent bz1-wx interval. This factor also did not increase recombination frequencies in the c1-sh1 and bz1-wx intervals, demonstrating independent control of recombination in adjacent intervals. Additional phenotypic analysis of recombination in the c1-sh1 and bz1-wx intervals and RFLP analysis of recombination along chromosomes 7 and 5 suggested that heritable factors controlling recombination in these intervals act largely independently and in trans. Our results show that recombination in these populations, and possibly maize in general, is controlled by both cis- and transacting factors that affect specific chromosomal regions.     

Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize

The 18 kDa high-methionine δ-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of δ-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine δ-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown.     

Immobilized microbes and a high-rate, continuous waste processor for the production of high Btu gas and the reduction of pollutants

Microbes capable of degrading organic wastes (sewage) are densely packed (immobilized) within the pores of controlled-pore ceramics. When the ceramic displays the optimum pore range for the reproduction of these microbes, the minimum volume for a very efficient reactor is required. A two-stage, anaerobic, immobilized microbe reactor has been designed, and laboratoryscale units have been constructed. A few of these units have been operated continuously for two years. These reactors were designed for the efficent conversion of carbon to methane in biodegradable molecules and for the effective transfer of that gas. The reactors were operated at 20, 30, and 40 degrees C at residence times of 2-5.5 h. The total chemical oxygen demand (COD) of the sewage varied from 800-2600 mg/L. The resulting gas contained greater than 90% methane and less than 5% CO(2). Approximately 32-54% of the influent total carbon was recovered as methane. The reduction in COD varied from 63 to 89%.     

Sequence analysis and comparison of cDNAs of the zein multigene family .

The nucleotide sequence of two zein cDNAs in hybrid plasmids A20 and B49 have been determined. The insert in A20 is 921 bp long including a 5' non-coding region of 60 nucleotides, preceded by what is believed to be an artifactual sequence of 41 nucleotides, and a 3' non-coding region of 87 nucleotides. The B49 insert is 467 bp long and includes approximately one-half the protein coding sequence as well as a 3' non-coding region of 97 nucleotides. These sequences have been compared with the previously published sequence of another zein clone, A30 . A20 and A30 , both encoding 19 000 mol. wt. zeins , have approximately 85% homology at the nucleotide level. The B49 sequence, corresponding to a 22 000 mol. wt. zein, has approximately 65% homology to either A20 or A30 . All three zeins share common features including nearly identical amino acid compositions. In addition, the tandem repeats of 20 amino acids first seen in A30 are also present in A20 and B49 .     

Learning enhancement and behavioral arousal induced by yohimbine

Learning of a food motivated delayed reinforcement autoshaping task was investigated in rats treated with water vehicle or the prototypical anxiogenic agent and alpha 2-adrenergic antagonist yohimbine (0.5 or 1.5 mg/kg, i.p. 30 min before behavioral testing). Unconditioned exploratory rearing activity was monitored concomitantly with acquisition of a lever touch response. The low dose of yohimbine enhanced learning, but it also increased unconditioned behavioral arousal. The high dose retarded acquisition, but when it was withdrawn the animals learned but exploratory activity increased beyond control levels prior to acquisition. Learning thus appeared to be related to the behavioral arousal produced by yohimbine, suggesting that learning enhancement by anxiogenic substances is not due to a direct effect on processes intrinsic to information storage and retrieval; rather, anxiogenic substances may be important modulators of vigilance and performance variables.     

Activation of Ras/Erk pathway by a novel MET-interacting protein RanBPM

MET is a receptor protein-tyrosine kinase (RPTK) for hepatocyte growth factor (HGF), which is a multifunctional cytokine controlling cell growth, morphogenesis, and motility. MET overexpression has been identified in a variety of human cancers. Oncogenic missense mutations of the tyrosine kinase domain of the MET gene have been identified in human papillary renal cell carcinomas. In this study, RanBPM, also known as RanBP9, is identified as a novel interacting protein of MET through yeast two-hybrid screen. RanBPM contains a conserved SPRY (repeats in splA and RyR) domain. We demonstrate that RanBPM can interact with MET in vitro and in vivo, and the interaction can be strengthened by HGF stimulation. RanBPM interacts with the tyrosine kinase domain of MET through its SPRY domain. We show that RanBPM can induce GTP-Ras association and Erk phosphorylation and elevate serum response element-luciferase (SRE-LUC) expression, indicating that RanBPM can activate the Ras-Erk-SRE pathway. We demonstrate that RanBPM, which itself is not a guanine exchange protein, stimulates Ras activation by recruiting Sos. On the cellular level, A704 cells, a human renal carcinoma cell line, transfected with RanBPM exhibit increased migration ability. Our data suggest that RanBPM, functioning as an adaptor protein for the MET tyrosine kinase domain, can augment the HGF-MET signaling pathway and that RanBPM overexpression may cause constitutive activation of the Ras signaling pathway.