Genomic and proteomic databases: large-scale analysis and integration of data.

With the completion of the human genome draft sequencing and assembly, an upcoming targeted goal will be identifying the proteins and changes in protein expression, which are derived from the genome template. There is important information already available on many proteins that can be a valuable resource to scientists in cardiovascular medicine as well as other fields. This article will summarize many of the different databases, their content and growth, and the differences between growth of nucleic acid and protein databases. Linkage between and integration of different protein and nuclei acid databases, along with related annotation, will greatly improve the information content and knowledge base with regards to protein data.

Virtual 2-D gel electrophoresis: visualization and analysis of the E. coli proteome by mass spectrometry.

Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis. This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated. Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage. Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains. Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels. In the 5.7-6.0 pH range, E. coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels. Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E. coli proteins.

Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity.

Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis.

We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E. coli. We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy. We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy. In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.

Resolution of genotypic and phenotypic properties of herpes simplex virus type 1 temperature-sensitive mutant (KOS) tsZ47: evidence for allelic complementation in the UL28 gene.

Herpes simplex virus type 1 (HSV-1) mutant tsZ47 was reported to be temperature sensitive for virus growth and transport of viral glycoproteins to the cell surface and to contain two different mutations (B. A. Pancake, D. P. Aschman, and P. A. Schaffer, (1983) J. Virol. 47,568-585). However, we found that similar amounts of glycoproteins B, C and H were expressed at the cell surface at the permissive and non-permissive temperatures and in addition, tsZ47 virus contained only a single mutation. UL28-null virus, gC delta 7B, failed to complement tsZ47 in mixed infections and tsZ47 replicated in UL28 but not gB transformed cell lines. A ts lesion of tsZ47 was mapped within a 1333 bp region of the UL28 gene by marker-rescue using overlapping DNA fragments. DNA sequencing identified a C to T transversion resulting in an R to W amino acid change at UL28 amino acid position 531. Southern Blot analysis and transmission electron microscopy demonstrated that tsZ47, is defective in cleavage and encapsidation of viral DNA. Mutant virus ts1203 (C. Addison, F.J. Rixon, and V.G. Preston, (1990) J. Gen. Virol. 71, 2377-2384) that contains a mutation in the 5' end of UL28, complemented tsZ47 in mixed infections. This suggests that allelic complementation may be occurring and UL28 may encode a protein with independently functioning domains, or that it participates in a multimer.