Reannotation of Shewanella oneidensis genome.

As more and more complete bacterial genome sequences become available, the genome annotation of previously sequenced genomes may become quickly outdated. This is primarily due to the discovery and functional characterization of new genes. We have reannotated the recently published genome of Shewanella oneidensis with the following results: 51 new genes have been identified, and functional annotation has been added to the 97 genes, including 15 new and 82 existing ones with previously unassigned function. The identification of new genes was achieved by predicting the protein coding regions using the HMM-based program GeneMark.hmm. Subsequent comparison of the predicted gene products to the non-redundant protein database using BLAST and the COG (Clusters of Orthologous Groups) database using COGNITOR provided for the functional annotation.

Set of novel tools for PCR primer design.

We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.

The promoter for tomato 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 has unusual regulatory elements that direct high-level expression.

The promoter region of tomato (Lycopersicon esculentum) 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 (HMG2) has been analyzed using the transient expression of HMG2-luciferase fusions in red fruit pericarp. The mRNA for HMG2 accumulates to high level during fruit ripening, in a pattern that coincides with the synthesis of the carotenoid lycopene. Unlike most promoters, the region that is upstream of the HMG2 TATA element is not required for high-level expression. The 180-bp region containing the TATA element, the 5' untranslated region, and the translation start site are comparable in strength of the full-length 35S cauliflower mosaic virus promoter. Pyrimidine-rich sequences present in the 5' untranslated leader are important in regulating expression. Also, the ATG start region has been found to increase translation efficiency by a factor of 4 to 10. An alternative hairpin secondary structure has been identified surrounding the HMG2 initiator ATG, which could participate in the translational regulation of this locus. HMG2 appears to be a novel class of strong plant promoters that incorporate unusual, positive regulators of gene expression.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.