Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-02-14
2003-09-23
Ponnaluri, Padmashri (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S069100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100
Reexamination Certificate
active
06623922
ABSTRACT:
FIELD OF THE INVENTION
The present invention comprises procedures for identifying, characterizing, and evolving cis-acting nucleic acid sequences that act in a cell-type specific manner to stimulate or repress the expression of linked genes or other neighboring sequences.
BACKGROUND OF THE INVENTION
A variety of cis-acting nucleic acid sequences influence expression levels of genes in prokaryotic and eukaryotic cells. These sequences act at the level of mRNA transcription, mRNA stability, or mRNA translation (Alberts B., Bray D., et al. (Eds.),
Molecular Biology of the Cell
, Second Edition, Garland Publishing, Inc., New York and London, (1989)). In the cases of RNA stability and translation, the cis sequences are present on the RNA molecules themselves. In the case of transcription, the cis sequences may be present either on the transcribed sequences or they may reside nearby in regions of the gene that are not transcribed.
In prokaryotes that have been studied, most of the transcriptional control sequences lie immediately upstream of the RNA start site in an area called the promoter. In the case of
E. coli
promoters, for example, the consensus promoter sequence consists of two regions, one located about 10 basepairs upstream of the start site, and one located about 35 bases upstream. These sequences coordinate the binding of RNA polymerase, the principal enzyme involved in transcription. Other sequences also influence the level of transcription of
E. coli
genes. These sequences include repressor-binding sites and other sites that bind ancillary factors that regulate interaction between RNA polymerase and the promoter.
In prokaryotes such as
E. coli
, little regulation is exerted at the level of transcript stability, probably because the cell division cycle is typically very short. Thus, transcript half-lives are generally only a few minutes. However, considerable control is exercised at the level of translation. In
E. coli
, sequences immediately upstream of the translational start site (Shine-Dalgarno sequences) mediate the binding of mRNA molecules to the ribosome, and hence, the efficacy of translation.
In eukaryotes, the control of gene expression is more complex but some of the same principles are involved. Gene expression levels are influenced not only by cis sequences that bind transcription regulatory factors, but also by sequences that affect the overall conformation of the DNA in the vicinity of the gene in question. These effects on chromatin structure are less well understood, but are likely to be very significant. It is thought that structural components such as histones and other proteins pack or unpack in a regulated fashion to affect the global and local conformations of DNA, and thus the accessibility of cis regulatory elements in or near genes.
The promoter regions of eukaryotic genes are also more complex than prokaryotic promoters and generally involve binding sites for numerous factors in addition to the RNA polymerase holoenzyme. Certain sequences are involved specifically in the process of transcription initiation, such as the TATA box (Myers R M, Tilly K, and Maniatis T.,
Science
232: 613-618 (1986)), whereas other sequences act to influence the rate of initiation. These latter sequences have been called enhancers, and they have the property of being relatively insensitive to position in the promoter (Wasylyk B., Wasylyk C., and Chambon P.,
Nucleic Acids Res
. July 25; 12: 5589-5608 (1984)). Many enhancers are located several kilobasepairs away from the gene whose expression level they regulate.
Because cell generation times in eukaryotes are typically longer than in prokaryotes, transcript stability is an important mode of regulation. For instance, some transcripts such as c-Fos have half lives on the order of minutes, while others have half lives on the order of hours. Sequences located at a variety of sites within the transcript influence the susceptibility of specific mRNA molecules to degradation by RNases within the cell (Ross J.,
Microbiol Rev
.: 423-450 (1995).
Translational regulation also plays a significant role in eukaryotic gene expression. Secondary structure in particular transcripts can influence translation rates, as can codon usages. In addition, the sequence composition surrounding the translational start site (the Kozak-consensus sequence) is an important factor in translational efficiency (Kozak M.,
Cell
January 31; 44: 283-292 (1986)).
In both prokaryotes and eukaryotes, the activity of many promoters is regulated according to the state of the cell. In metazoans, the situation can be much more complex because certain promoters may be active only in specific cell lineages. Thus, their activity must be regulated according to the particular time in development of the organism and the specific cell type.
Genetic screens and selections allow identification of regulatory elements in genes. If a powerful genetic selection or screen is enforced on a population of cells, it is possible to identify variants that have properties worthy of further study. Multiple rounds of selection or screening may permit the ultimate identification of variants in cases where a single round of selection/screen is not sufficient to enrich the population of desired variants. Genetic selections typically involve conditions whereby wild type cells die or grow slowly compared to variant cells in the population. Such conditions may be forced upon a culture of cells or a population of organisms. An equivalent process may involve a “screen and pluck” approach, where interesting variants are identified from the population, separated, and allowed to replicate in isolation. Such a process ultimately leads to an enrichment in the selected population for variants with the desired phenotypic traits, and a diminution of cells or organisms with the parental phenotype.
Numerous approaches have been applied to the identification and study of cis regulatory sequences. However, in general the approaches have been relatively labor intensive and slow. In addition, the approaches have generally been aimed at the study of the behavior of cis sequences in the natural setting; i.e., the intention has been to study the normal regulation of such sequences in the cell.
In certain cases, cis sequences have been deliberately engineered to control expression of particular genes in desirable ways. For example, it is useful to regulate tissue specificity and levels of exogenous genes using defined regulatory elements. This may involve fine control over tissue specificity, e.g., as in expression of the SV40 T antigen (TAg) in pancreatic islet beta cells by linking the TAg gene to the insulin promoter (Hanahan D.,
Nature
May 11; 20: 2233-2239 (1985)), or it may involve efforts to maximize expression, e.g., as in the use of viral regulatory sequences such as the CMV enhancer (Wilkinson G. W., and Akrigg A.,
Nucleic Acids Res
. May 11; 20: 2233-2239 (1992)), or it may involve efforts to modulate expression levels from low to high, e.g., as in the LacSwitch (Fieck A., Wyborski D. L., and Short J. M.,
Nucleic Acids Res
. 20: 1785 (1992)) and TetSwitch systems (Iida A., Chen S. T., et al.,
J. Virol
70: 6054-6059(1996)).
A variety of techniques have been used in to identify cis sequences that regulate gene expression. These include biochemical methods that identify sites of interaction with protein factors, comparative sequence analysis, characterization of regulatory mutations in genes, and assay of deliberately constructed sequence variants for their effects on gene expression (Latchman David S.,
Eukaryotic Transcription Factors
Second Edition, Academic Press, London (1996); McKnight S. L., and Yamamoto K. R. (Eds.),
Transcriptional Regulation
, CHSL Press, New York (1992)). Such methods have the drawback that they often require some a priori knowledge of the nucleic acid sequence of the regions of interest. In addition, several methods have been employed to “trap” cis sequences that have promoter activity. In prokaryotes, this often involves insertion of reporter construct
Caponigro Giordano M.
Kamb Carl Alexander
Deltagen Proteomics
Fenwick & West LLP
Ponnaluri Padmashri
LandOfFree
Methods for identifying, characterizing, and evolving... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for identifying, characterizing, and evolving..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for identifying, characterizing, and evolving... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3034831