Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-10-06
2003-01-28
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120, C435S091200, C435S069100
Reexamination Certificate
active
06511832
ABSTRACT:
BACKGROUND OF THE INVENTION
At least some of the present invention may have been made with funds from the United States Government, which may therefore have certain rights in this invention.
1. Field of the Invention
Aspects of the present invention relate to the discovery and use of baculovirus RNA polymerase for the production of capped and polyadenylated transcripts in vivo and in vitro. Aspects relate to useful tools and techniques for biotechnologists, including more stable RNA transcripts produced.
2. Background of the Related Art
A. Baculovirus
Baculoviruses are popular eukaryotic expression vectors. The baculovirus system has been used to produce hundreds of different proteins for basic research and pharmaceutical applications such as medical therapeutics, diagnostics, vaccines, and drug discovery. Baculoviruses have been adapted as expression vectors because they normally produce abundant amounts of a viral protein called polyhedrin during the very late stage of viral infection. Polyhedrin is essential for the propagation of baculoviruses in insects, but is nonessential for growth in tissue culture. Thus the polyhedrin open reading frame can be replaced by coding regions for target genes of choice, and in many cases, target proteins are expressed at levels equivalent to that of polyhedrin, which is approximately 1 mg per ml of culture. In addition, eukaryotic proteins are frequently subject to the appropriate post-translational modifications, including phosphorylation, glycosylation, and acylation.
Autographa californica
nuclear polyhedrosis virus (AcNPV) is the prototype member of the Baculoviridae, which is a large family of DNA viruses that are pathogenic for invertebrates. The AcNPV genome consists of a double-stranded, supercoiled DNA molecule of 134 kbp, and potentially encodes 150 proteins (Ayres et al., 1994). In infected cells, AcNPV genes are expressed in a temporally controlled and ordered fashion (Blissard and Rohrmann 1990; O'Reilly et al., 1992). Viral genes are classified as early or late based on their requirements for viral DNA replication. Transient expression assays suggest that there are two distinct classes of early genes. One class includes genes like ie1 and ie2 that are highly expressed in the absence of other viral proteins and enhancer elements (Guarino and Summers, 1986a; Carson et al., 1988). The other class includes genes like 39k that are expressed at basal levels in the absence of viral factors, but whose expression is enhanced approximately 1000-fold in the presence of IE1 and cis-linked enhancer elements (Guarino and Summers, 1986b). Baculovirus early promoters resemble those transcribed by eukaryotic RNA polymerase II (pol II). Transcription of the early genes is inhibited by &agr;-amanitin, consistent with the hypothesis that early genes are transcribed by host pol II (Fuchs et al., 1983; Grula et al., 1981). Transcription of several early baculovirus genes initiates within a conserved ‘CAGT’ motif. Mutagenesis of this element was shown to affect transcription initiation in the 39k promoter (Guarino and Smith, 1992) and in the gp64 promoter (Blissard et al., 1992), suggesting that CAGT functions as an initiator element.
Late genes are also divided into two classes: the late genes, many of which encode viral structural proteins, and the very late genes, which are associated with the formation of viral occlusions. Transcription of both classes of late genes is resistant to (&agr;-amanitin (Huh and Waver, 1990), suggesting that these genes are transcribed by a viral-encoded RNA polymerase. This polymerase may be encoded by the lef-8 gene of AcNPV (Passarelli et al., 1994). Late and very late genes contain the consensus late promoter element, TAAG. This core element appears to function as both a promoter and an mRNA start site (Rankin et al., 1988).
Several proteins (LEF=late expression factor) required for late gene expression have been identified (Table 1; Passarelli and Miller, 1993a,b,c,1994; Passarelli et al., 1994; Li et al., 1993; Morris et al., 1994; Lu and Miller, 1994; McLachlin and Miller, 1994). The genes encoding these proteins were mapped using a transient transfection assay. Some of these proteins may be directly involved in late gene expression, and some may only be required for earlier events in the virus life cycle. For example, IE1 and IE2 transactivate early gene expression (Guarino and Summers, 1986a; Carson et al., 1988); and LEFs 1-3, helicase, and DNA polymerase are required for DNA replication (Kool et al., 1994). Little is known about the roles of LEFs. The predicted amino acid sequence of LEF-8 contains a motif that is conserved in RNA polymerases from various sources (Passarelli et al., 1994).
TABLE 1
Viral proteins required for the expression of late and very late
genes.
Molecular
Protein
weight
Structural features
Associated functions
IE1
71 kDa
Acidic region and DNA
Transactivates early
binding domain
genes. Binds to
enhancers/origins
1E2
47 kDa
Several motifs common
Co-activator for early
to transcription factors
transcription
LEF-1
31 kDa
Nucleotide triphosphate
Required for DNA
binding motif
replication
LEF-2
23 kDa
Required for DNA
replication
LEF-3
44 kDa
Required for DNA
replication Single-
stranded DNA
binding protein
Helicase
143 kDa
Homology with helicases
ts mutants are DNA
negative. Required
for DNA replication.
DNA pol
114 kDa
Homology with DNA
Required for DNA
polymerases
replication
P35
35 kDa
Suppressor of
apoptosis
LEF-4
54 kDa
LEF-5
31 kDa
LEF-6
20 kDa
LEF-7
24 kDa
Zinc finger
LEF-8
102 kDa
RNA polymerase motif
LEF-9
59 kDa
LEF-10
9 kDa
pp31/39K
31 kDa
DNA binding protein
VLF-1
44 kDa
Homology with
Very late expression
integrases
factor
B. In Vitro Transcription of Capped RNAs
A number of research protocols have been developed that require milligram amounts of purified mRNAs. These RNAs are usually transcribed in vitro using plasmids or PCR fragments as templates. The most commonly used RNA polymerases for in vitro transcription are the enzymes encoded by the bacteriophages T3, T7, and SP6. These RNA polymerases are useful because they are highly active, very processive enzymes that recognize a specific promoter. In vitro transcription reactions with these enzymes are usually done as run-off assays. In this type of assay, the plasmid DNA is linearized prior to transcription, and so the 3′-end of the message is determined by the end of the DNA template. As a result, the transcripts produced are appropriate for translation in prokaryotic systems, but are not optimal for eukaryotic systems because they are not processed at the 5′ end with a 5′-methyl-7-guanosine cap and at the 3′ end with a poly(A) tail.
The most common use of in vitro transcribed RNAs is cell free translation, either in rabbit reticulocyte or in wheat germ extracts. Efficient in vitro translation of the RNAs requires the presence of a 5′-methyl-7-guanosine cap. The cap structure is important for binding of ribosomes to the RNA and also for message stability. Thus uncapped transcripts, which are the normal product of bacteriophage RNA polymerases, are not efficiently translated in eukaryotic cell free systems. Other protocols that require capped transcripts include microinjection of mRNAs into oocytes or transfection of mRNAs into animal cells or plant cells for the purpose of studying in vivo RNA processing, RNA transport, or protein function. Capping for these in vivo is essential because RNAs that are uncapped are rapidly degraded by cellular RNases. Also, in vitro splicing assays and the characterization of splicing factors require capped RNAs as substrate because the cap structure helps to target splicing enzymes to their substrates.
The production of capped mRNAs is usually done by adding cap analog (7mGpppG) to the in vitro transcription reactions. Cap analog can be incorporated in place of GTP al the 5′ end of the message. Although this is not the typical route of cap formation, the use
Dong Wen
Guarino Linda A.
Jin Jianping
Fulbright & Jaworski L.L.P.
Jones W. Gary
Taylor Janell E.
Texas A&M University System
LandOfFree
In vitro synthesis of capped and polyadenylated mRNAs using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with In vitro synthesis of capped and polyadenylated mRNAs using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and In vitro synthesis of capped and polyadenylated mRNAs using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3001845