Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-05-03
2003-05-06
Guzo, David (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S069100, C435S320100, C435S325000, C435S468000, C435S410000, C435S419000, C536S023100, C536S023400, C536S023720, C536S024100, C800S295000
Reexamination Certificate
active
06559359
ABSTRACT:
FIELD OF INVENTION
The present invention relates generally to retroviruses, pro-retroviral polynucleotides including pro-retroviral DNA, pro-retroviral-like DNA and more specifically to recombinant vectors derived therefrom for use in delivering genetic information to susceptible target plant cells.
BACKGROUND OF INVENTION
Repetitive DNA sequences are a common feature of the genomes of higher eukaryotes. Repetitive DNA family members in animals and higher plants are tandemly repeated or interspersed with other sequences (Walbot and Goldberg, 1979; Flavell, 1980), and may constitute more than 50% of the genome (Walbot and Goldberg, 1979). Estimates of the proportion of repetitive DNA in the soybean genome range from 36% to 60% (Goldberg, 1978; Gurley et al., 1979).
High copy-number repeats on the order of 10
5
per haploid genome comprise only 3% of the soybean genome, whereas moderately repetitive sequences with copy-numbers in the 10
3
range occupy 30-40% of the genome (Goldberg, 1978). Electron micrographic examination of these moderately repetitive sequences demonstrate that they average about 2 kb in length; however, 4% of those observed exceed 11 kb (Pellegrini and Goldberg, 1979).
Most of the highly repetitive sequences in higher eukaryotic genomes are relatively short and are organized in tandem arrays. For example, the chromosomal region adjacent to the centromere in higher eukaryotes is composed of very long blocks of highly repetitive DNA, called satellite DNA, in which simple sequences are repeated thousands of times or more. Tandemly repeated elements found in the soybean genome also include the ribosomal RNA (rRNA)-encoding genes. The approximately 800 rDNA copies are organized as one or more clusters of tandemly repeated 8-kb or 9-kb units (Friedrich et al., 1979; Varsanyi-Breiner et al., 1979).
The genomes of most higher eukaryotes also contain highly repetitive sequences that are distributed evenly throughout the genome, interspersed with longer stretches of unique (or moderately repetitive) DNA. These interspersed repetitive DNA elements are variable in length, are recognizably related but not precisely conserved in sequence, and exhibit relatively small repeat frequencies (Lapitan, 1992).
The dispersal pattern of interspersed repetitive elements in higher eukaryotic genomes has led to the suggestion that they are, or once were, transposable elements known as transposons (Flavell, 1986; Lapitan, 1992). Transposons are genetic elements that can move from one chromosomal location to another, without necessarily altering the general architecture of the chromosomes involved. The existence of transposons has only found general acceptance within the last few decades. Genes were originally believed to have fixed chromosomal locations that only change as a result of chromosomal rearrangements resulting from illegitimate crossing-over between incompletely homologous short sections of DNA. Then, in the late 1940's, McClintock's pioneering experiments with maize showed that certain genetic elements regularly “jump”, or transpose, to new locations in the genome (McClintock, 1984).
Transposable elements (TEs) reside in the genomes of virtually all organisms (Berg and Howe, 1989). TEs encode enzymes that bring about the insertion of an identical copy of themselves into a new DNA site. Transposition events involve both recombination and replication processes that frequently generate two daughter copies of the original transposable element; one remains at the parental site, while the other appears at the target site (Shapiro, 1983).
Two major classes of eukaryotic TEs have been identified, which are distinguished by their mode of transposition (Finnegan, 1989). Class I elements transpose via the creation of an RNA intermediate that is then re reverse-transcribed to create a DNA copy that integrates at the target site. This class includes several families of retroelements—retrotransposons and retroviruses—including the copia elements of
Drosophila melanogaster,
the gypsy/Ty3 family, the Ty1 element of yeast, and the mammalian immunodeficiency and Rous sarcoma (RSV) retroviruses. Each of these retroelement families are characterized in part by the presence of long terminal repeats (LTRs) at their borders (Finnegan, 1989); however, this class also includes non-LTR-containing elements like Cin4 from maize (Schwarz-Sommer and Saedler, 1988) and the mammalian L1 family (Hutchinson et al. 1989).
The copia elements in
D. melanogaster
possess long terminal direct repeats. There are more than 11 families of copia-like elements; the members of each are well-conserved and are located at 5 to 100 different sites in the Drosophila genome. These elements are about 5000 base pairs (bp) long, with long terminal repeats (LTRs) several hundred bp in length that vary in both sequence and length between families. At the termini of each element are short imperfect inverted repeats of about 10 bp.
Insertion of copia into a new chromosomal site is accompanied by replication of a 3-6 bp stretch of target DNA; the length, but not the sequence, of the direct repeats that consequently appear immediately before and after the element is the same for all members of the same family. Copia elements have one long open reading frame (ORF) that encodes proteins homologous to those of RNA tumor viruses: homologies to reverse transcriptase, integrase, and nucleic acid-binding proteins suggest that these proteins function to create an RNA intermediate for copia transposition.
Class II elements, like the
Drosophila melanogaster
P element (Engels, 1989; Rio, 1990) and the maize Ac/Ds element (Federoff, 1989), transpose directly to new sites without the formation of an RNA intermediate. P elements reside at multiple sites in the Drosophila genome and are 0.5 to 1.4 kb in length, bounded by perfect inverted repeats of 31 bp. They represent internally deleted versions of a larger element of about 3 kb called a P factor, which occurs in one or a few copies only in so-called “P strains” of Drosophila. Upon insertion into a new site in the genome, P elements create 8 bp duplications of the target sequence.
The Ac/Ds system in maize consists of Ds elements, which, like the P elements of Drosophila, are derived from a larger complete element called Ac. Ds elements exist in several different lengths, from 0.4 to 4 kb. Unlike P elements, Ds elements remain stationary within the chromosome unless an Ac element is also present. Ds elements contain perfect inverted repeats of 11 bp at their termini, flanked by 6-8 bp direct repeats of the target DNA. When a Ds (or Ac) element transposes, it leaves behind imperfect but recognizable duplications of the 6-8 bp target sequence.
As stated above, it appears likely that many interspersed repetitive DNA families are, or once were, transposons. In soybean, an interspersed repetitive DNA family whose structural characteristics clearly define it as a transposon family is the Tgm family. The Tgm family is related to the maize En/Spm transposons and consists of fewer than 50 members ranging in size from under 2 kb to greater than 12 kb (Rhodes and Vodkin, 1988).
Retroviruses are type I transposons consisting of an RNA genome that replicates through a DNA intermediate. Although the viral genome is RNA, the intermediate in replication is a double-stranded DNA copy of the viral genome called the provirus (Watson et al., 1987). The provirus resembles a cellular gene and must integrate into host chromosomes in order to serve as a template for transcription of new viral genomes (Varmus, 1982). New genomes are processed in the nucleus by unmodified cellular machinery.
The viral genome RNA looks like a cellular messenger RNA (mRNA), but does not serve as such following infection of a cell. Instead, an enzyme called reverse transcriptase (which is not present in the cell, but is instead carried by the virion) makes a DNA copy of the viral RNA genome, which then undergoes integration into cellular chromosomal DNA as a provirus. Integration of the viral DNA is precise with respect to
Guzo David
Loyola University of Chicago
Marshall Gerstein & Borun
LandOfFree
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