Paul V. Allen

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.

Partial amino acid sequence of potato solanidine UDP-glucose glucosyltransferase purified by new anion-exchange and size exclusion media

Solanidine UDP-glucose glucosyltransferase (SGT) is involved in the biosynthesis of steroidal glycoalkaloids in potatoes. This enzyme is present at an extremely low level, is inherently unstable, and copurifies with the major storage protein patatin during isolation. We describe an improved method for isolating SGT from greening potato peel using two new chromatographic supports, Macro-Prep 50 Q anion-exchange and Superdex 75HR size exclusion media, under medium-pressure conditions at room temperature. The enzyme preparation was further resolved by SDS-PAGE and the proteins transferred to PVDF membrane (Immobilon-P). Two protein bands corresponding to active forms of SGT (36 and 37 kDa) were excised and cleaved with cyanogen bromide in trifluoroacetic acid. The resultant peptide mixtures were then separated by Tricine-SDS-PAGE and transferred to a PVDF membrane (Pro-Blott). The two major peptide bands observed in both digests (17 and 19 kDa) were sequenced. Identical N-terminal sequences were obtained from the 19-kDa peptides from both digests.

Structure of Two Solanum bulbocastanum Polyubiquitin Genes and Expression of Their Promoters in Transgenic Potatoes

Two polyubiquitin genes, bul409 and bul427, were isolated from a Solanum bulbocastanum BAC library. The bul409 and bul427 genes encode hexameric and heptameric polyproteins, respectively. bul427 exhibits a number of features suggestive of a pseudogene: (1) The last ubiquitin monomer of bul427 is interrupted by a frame shift mutation. (2) The coding sequence is flanked 3′ by mitochondrial and chloroplast sequences and 5′ by a protein kinase pseudogene. However, characterization of cDNAs amplified using bul427-based primers demonstrated that bul427 is transcriptionally active. Chimeric transgenes encoding β-glucuronidase (GUS) translationally fused to the first ubiquitin-coding units of bul409, a truncated version 409s, and bul427 were constructed and introduced into potato. In transgenic potato lines both S. bulbocastanum promoters were weakly transcribed in tubers and efficiently transcribed in leaves. In leaves, bul409s was wound-induced, while bul427 was not. In tubers both promoters were wound-induced. In unwounded leaves and tubers, the steady state mRNA levels from both bul promoters were lower than the steady state mRNA levels from the Cauliflower Mosaic Virus 35S promoter. However, in the leaves and tubers of many of the transgenic lines the GUS activity was significantly higher in the bul lines compared to the 35S lines. The apparent inconsistency of higher enzymatic activity correlated with lower steady state levels of mRNA demonstrates the enhanced protein expression observed with ubiquitin fusion proteins.ResumenDos genes de poliubiquitina, bul409 y bul427, fueron aislados de una librería BAC de Solanum bulbocastanum. Los genes bul409 y bul427 codifican poliproteínas hexámera y heptámera, respectivamente. El bul427 exhibe varios rasgos que sugieren un seudo gen: (1) El último monómero de bul427 es interrumpido por una mutación con desplazamiento de la pauta de lectura. (2) La secuencia codificante es flanqueada en 3′ por secuencias mitocondriales y de cloroplastos y en 5′ por un seudogen de proteína quinasa. Sin embargo, la caracterización de cDNAs amplificadas usando iniciadores basados en bul427 demostró que bul427 es transcripcionalmente activo. Se construyeron e introdujeron en papa transgenes quiméricos que codifican β-glucuronidasa (GUS) translacionalmente fusionados a las primeras unidades que codifican ubiquitina de bul409, una versión truncada de 409s y bul427. En las líneas de papa transgénica de S. bulbocastanum, los promotores fueron débilmente transcritos en los tubérculos y eficientemente transcritos en hojas. En hojas el bul409s fue inducido por heridas, mientras que bul427 no fue. En tubérculos, ambos promotores fueron inducidos por heridas. En hojas y tubérculos sin herir, los niveles mRNA en estado de equilibrio para ambos promotores bul fueron más bajos que los niveles de mRNA en estado de equilibrio del promotor 35S del virus del Mosaico de la Coliflor. Sin embargo, en hojas y tubérculos de muchas líneas transgénicas, la actividad de GUS fue significativamente más alta en las líneas bul en comparación con las 35S. La aparente inconsistencia de una actividad enzimática mayor, correlacionada con bajos niveles de mRNA en estado de equilibrio demuestra el incremento de la expresión de proteína observada con proteínas fusión ubiquitina.

Use of hair as an indicator of environmental lead pollution in women of child-bearing age in Karachi, Pakistan and Bangladesh.

The present study was prompted by an observation made during a visit to Karchi in 1986 when three blood samples from two women and a man, all between 30-35 years yielded blood Pb levels of 31.4, 21.9 and 41.7 ug Pb/dl blood. Karachi is a highly industrialized urban center with very large numbers of motor vehicles, all of which use leaded gasoline. Lead-based paint is also extensively used in the city. The present study was conducted to determine if hair metal analyses could be used to determine elevated exposures to lead in women of child-bearing age living in Karachi (Pakistan). Rural Bangladeshi women living in areas with little or no vehicular traffic and no factories in the vicinity were used as controls.