A Tobacco-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice

Thymosinβ4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bpTβ4gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify4×Tβ4expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression inEscherichia coliat promoting splenocyte proliferationin vitroand wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

Characterization of a Maize Chromosome 4 Centromeric Sequence: Evidence for an Evolutionary Relationship With the B Chromosome Centromere

Previous work has identified sequences specific to the B chromosome that are a major component of the B centromere. To address the issue of the origin of the B and the evolution of centromere-localized sequences, DNA prepared from plants without B chromosomes was probed to seek evidence for related sequences. Clones were isolated from maize line B73 without B chromosomes by screening DNA at reduced stringency with a B centromeric probe. These clones were localized to maize centromere 4 using fluorescence in situ hybridization. They showed homology to a maize centromere-mapped sequence, to maize B chromosome centromere sequences, and to a portion of the unit repeat of knobs, which act as neocentromeres in maize. A representative copy was used to screen a BAC library to obtain these sequences in a larger context. Each of the six positive BACs obtained was analyzed to determine the nature of centromere 4-specific sequences present. Fifteen subclones of one BAC were sequenced and the organization of this chromosome 4-specific repeat was examined.

Cloning of a DNA repeat element from horse: DNA sequence and chromosomal localization

A DNA repeat element, revealed initially by digestion of horse DNA with TaqI, was cloned and characterized by Southern and in situ hybridization studies and nucleotide sequencing. The clone, e4/1, consisted of 32 tandem reiterations of a unit repeat of 21–22 bp, and produced multilocus DNA fingerprinting profiles that were useful for parentage analysis in horses. The tandem repeat element was shown by in situ hybridization to be localized in the centromeres of the acrocentric but not metacentric classes of horse chromosomes.Key words: Equidae, Equus caballus, DNA repeat, DNA fingerprinting, centromere.

Cloning and Expression of a Synthetic Mussel Adhesive Protein in Escherichia Coli

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were cloned and expressed in Escherichia coli. The bioadhesive precursor (BP, Mx−25,000) was expressed from one 600-bp gene comprised of a 30-bp unit repeat. The repetitious gene appeared stable in a T7-based host/vector system.Using the T7 expression system for induction, BP was produced at levels approaching 60% of total cell protein. BP was found both in intracellular inclusions and in the soluble fraction. Interestingly, methionine was processed from the N-terminus of the purified protein to give an authentic consensus precursor protein.