Proteins--Synthesis

The proteolytic activities of the ADAM (a disintegrin and metalloproteinase),
ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and
MMP (matrix metalloproteinase) protein families play important roles in normal and
multiple pathological conditions. These metalloproteases have potential implications
in the degradation of the extracellular matrix and in the processing of bioactive
molecules. Under pathological conditions these proteases are involved in many
diverse processes from tumor cell migration to cartilage destruction in rheumatoid
arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs,
and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from
multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic
normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared
between normal and metastatic states, and also between tissue and cultured cells.
Substantial differences have been observed in the level of ADAM and MMP mRNA
expression between tissue and cell lines. In general, the level of expression is several
folds higher in cultured cells compared to the isogenic tissue they are derived from.
Protein microarrays were utilized in order to evaluate the correlations between MMP
and TIMP mRNA copy numbers and protein abundance in cell culture. In several
cases, distinct differences were observed regarding the localization of the proteins
examined. In order to determine if the metalloprotease genes that were elevated at the
level of RNA expression produce functional proteins, the foundations of an in situ
FRET assay have been established. This will greatly aid in a better understanding of
the behavior of metallopeptidases in a cellular context.

tmRNA is a small stable RNA present in Eubacteria. Through a mechanism called
trans-translation, tmRNA mediates ribosome rescue and quality control of proteins and
mRNA. In this study, the Escherichia coli (E. coli) mutant lacking tmRNA was
demonstrated hypersensitive to oxidative stress. The role of tmRNA-mediated
surveillance mechanism in protecting E. coli cell under oxidative stress condition was
examined. The tmRNA-mediated tagged protein levels were elevated in cells under
oxidative stress condition, demonstrating the enhanced need for tmRNA under such
condition. Our results suggest that mRNA damage by oxidative stress may cause reduced
cell viability, and that tmRNA is required to rescue cells under such condition.
Furthermore, our observations showed that tmRNA is required for the optimal growth of
E. coli under normal aeration but not under anaerobic condition, suggesting that oxidation
ofmRNA is the major reason for requirement oftmRNA during normal aeration.

The Bel family of genes are fundamental to the apoptotic mechanism. Bcl-x a
member of this family, is alternatively spliced to create two main isoforms a long
(Bcl-xL) and a short (Bcl-xS) variant. The long form exhibits anti-apoptotic activity,
while the short form favors apoptosis. The proper balance of expression of these two
isoforms is crucial for several developmental processes such as thymic selection and
neural reshaping. A number of cancer types have been shown to over-express the long
form, thereby granting them some protection from apoptosis. To study the
transcriptional and post-transcriptional mechanisms regulating gene expression, the
Bcl-x gene has been utilized. A complex mini-gene construct has been create in order
to monitor the effects that promoter sequences, 5'UTR and 3'UTR's have on mRNA
splicing, RNA export, stability and translation. Abundant evidence exists indicating
that RNA processing events such as transcription, splicing and export are coupled, yet
the mechanisms and factors involved in regulating these processes are poorly
understood. The mini-gene is identical to the endogenous gene with the exception of a deletion to the 50Kb intron and the addition of a tag to differentiate the mini-gene
product from the endogenous mRNA and protein. This novel system allows for the
study of transcriptional and post-transcriptional mechanisms regulating gene
expression from RNA biogenesis on to the protein level.