Polymerase I promoter plasmid and vector constructs

Details Polymerase I promoter plasmid and vector constructs Polymerase I promoter plasmid and vector constructs

1992-03-04

2002-04-09

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S325000, C435S353000, C435S366000, C536S023100, C536S024100

Reexamination Certificate

active

06368862

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to molecular biology and particularly to plasmid and vector constructs for obtaining RNA polymerase I-specific expression of a nucleotide sequence in a mammalian cell.
BACKGROUND OF THE INVENTION
Transcription in mammalian cells is initiated at promoter elements in the 5′ region of a gene by polymerase binding at specific transcription initiation nucleotide sequences. The determination of which RNA polymerase, e.g., polymerase I, II, or III, will transcribe a given gene is also made at these specific promoter sequences. RNA polymerase II transcribes mRNA at many chromosomal loci, while several small structural RNAs are transcribed by RNA polymerase III. RNA polymerase I (Pol I) appears specific for ribosomal DNA (rDNA) promoter sequences that direct transcription of ribosomal RNA (rRNA).
mRNA transcribed from a chromosomal gene by RNA Polymerase II (Pol II) has the following significant structural features: namely, 1) a 5′ trimethyl G “cap”; 2) an AUG codon in the proper sequence context to allow ribosomal initiation of translation; and, 3) a 3′ poly A tail. The trimethyl G “cap” is thought to be necessary to allow ribosomes to recognize a transcript as mRNA. Ribosomes initiate translation at the “capped” 5′ end of an mRNA, by scanning along the transcript until they encounter the first AUG codon in the proper sequence context. The 3′ poly A tail of an mRNA is thought to contribute stability to an mRNA.
rRNA transcription differs from mRNA in noteworthy ways. rRNA is encoded from several hundred tandemly repeated copies of the ribosomal genes (rDNA) that are located in five different clusters at various chromosomal loci in mammalian cells. rDNA is also located in structural elements in the nucleus referred to as the nucleolar organizing center. Nucleoli form at this site when rDNA is actively being transcribed into rRNA, and a large cluster of nascent ribonucleoprotein particles forms around each active locus. These clusters are the structures visible in the light microscope that have been called “nucleoli.” Processing of a ribosomal precursor rRNA (e.g., from a 28S precursor to 18S and 5.8S subunits) occurs in the nucleoli, and ribosomal proteins also associate with the rRNA before rRNA is transported from the nucleus to the cytoplasm as ribosomes. In addition, rRNA does not perform the functions of mRNA and is not translated by ribosomes into protein. Instead, rRNA is assembled into ribosomal proteins to form ribosomes which play a role in protein synthesis.
Features of Pol I transcription are notably different from the transcription of chromosomal genes by Pol II. For instance, an rRNA transcript synthesized by Pol I does not receive a trimethyl G “cap,” instead, Pol I-specific transcripts retain a simple triphosphate at the 5′ end and thus may not be recognized as mRNA by ribosomes. rRNA encoded by Pol I also has no AUG codons in a proper context, and rRNAs do not receive a 3′ polyA tail. Current understanding of Poll transcription of rRNA initiated from an rDNA promoter has been reviewed recently (Reeder, R. H. 1991. In:
Transcriptional Regulation
, K. Yamamoto and S. L. McKnight, Eds, Cold Spring Harbor, N.Y., in press).
Promoters specific for RNA polymerase II are associated with chromosomal genes, and many such Pol II promoters have been isolated and used in expression plasmids and vectors. Several reports also describe experiments attempting to use a Pol I rDNA promoter to drive mRNA expression. Fleisher and Grummt (1; see the appended Citations) reported a mouse rDNA promoter attached to a gene for SV40 T-antigen, and Grummt and Skinner (2) reported a construct with a mouse rDNA promoter linked to a chloramphenicol acetyltransferase (CAT) marker gene. In the latter case, analysis of total cellular RNA reportedly showed high levels of transcript RNA, but only low levels of CAT activity were reportedly observed. A mouse rDNA promoter was also reportedly used by Surmacz et al. (4) to drive the SV40 T-antigen gene, but again the level of expression obtained was reportedly 5-to 10-fold poorer than that obtained with a Pol II promoter. Smale and Tijan (3) reportedly fused a human rDNA promoter to a gene for thymidine kinase (TK) marker gene, and although large amounts of transcripts were reportedly observed the RNA transcripts were reportedly unstable, not polyadenylated, and found largely in the nucleus rather than in the cytoplasm. Also, when the rDNA promoter was intentionally damaged in the latter study, i.e., by partial deletion of upstream sequences, it was revealed that transcription was initiated at two “cryptic” Pol II promoter sites in the plasmids. The finding of cryptic Pol II initiation sites raises the possibility that transcription attributed to rDNA promoters and Pol I may instead be mediated by Pol II transcription initiated at cryptic Pol II promoters. Lopata et al. (6) reported similarly that a mouse rDNA promoter driving a CAT gene expressed only low levels of CAT enzyme activity, and the only polysome-associated RNA reportedly came from aberrant initiation attributable to Pol II. The latter two reports appear to caution that extra care is needed to show that mRNA or protein expression is Pol I-specific, rather than mediated by another RNA polymerase initiating transcription at a cryptic promoter site(s). Thus, while rDNA promoter plasmids reportedly produced relatively large quantities of nuclear RNA transcripts, the transcripts remained in the nucleus and were inefficiently translated into protein product.
SUMMARY OF THE INVENTION
The invention provides rDNA promoter constructs useful in plasmids and vectors that direct transcription of RNAs in a Pol I-specific manner. The efficiency of protein production from the resulting transcripts approaches and in some cases surpasses that achieved with a strong Pol II-specific promoter. The nucleic acid constructs of the invention offer several advantages over previous Pol II vector systems, because rDNA promoters are highly active in essentially all cell types, and Pol I expression is under different cellular controls during growth and development than Pol II. The subject constructs provide quantitatively higher expression levels than achievable with previous Pol I constructs, and furthermore provide opportunities for constitutive expression.
The invention is embodied by constructs having four elements in a serial array: namely, a first nucleotide sequence, capable of hybridizing under stringent conditions to an rDNA promoter element; a second nucleotide sequence, capable of hybridizing under stringent conditions to an internal ribosome entry signal (IRES); a third nucleotide sequence containing a coding region of interest; and a fourth nucleotide sequence containing a polyadenylation (polyA) signal sequence. Pol I-specific expression of the second, third, and fourth nucleotide sequence is achieved by introducing the construct into a permissive cell. The latter process provides Pol I-specific expression levels that are higher than the levels achievable with an rDNA promoter construct lacking the IRES and polyA elements. The invention also provides genetically engineered stable cell lines that contain the subject constructs and that express the coding region third nucleotide sequence in a Pol I-specific manner. Pol I-specific expression can be verified by testing: a) that the expression is not substantially inhibitable by &bgr;-amanitin (i.e., an inhibitor of Pol II and Pol III); or, b) that the transcript polysome-associated RNA from the construct is 5′-trimethyl-Guanine-cap-deficient RNA; and/or c) that the expression is species-specific, e.g., a human rDNA promoter is expressed in human but not rodent cells, and a rodent rDNA promoter is expressed in rodent cells but not in human cells.
The subject constructs may be provided with a fifth element located upstream from the rDNA promoter element in the serial array. This fifth element is capable of hybridizing under stringent conditions to an rDNA enhancer e

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