Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-14
2002-07-16
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S183000, C435S412000, C536S023100, C536S023500, C536S024310, C536S024330, C800S278000, C800S295000
Reexamination Certificate
active
06420117
ABSTRACT:
BACKGROUND
Transposable elements are divided into two classes: Class 1, or retro-elements include the most abundant element in plants, the long terminal repeat (LTR) retrotransposons (such as Tnt1, Opie, Huck, and BARE1) and also the long interspersed nuclear elements (LINEs, also known as non-LTR retrotransposons), and short interspersed nuclear elements (SINEs). For all Class 1 elements, it is the element-encoded mRNA, and not the element itself, that forms the transposition intermediate. In contrast, Class 2 or DNA elements are characterized by short terminal inverted repeats (TIRs) and, most importantly, transposition via a DNA intermediate. Plant DNA elements (such as Ac/Ds, Spm/dSpm and Mutator) usually excise from one site and re-insert elsewhere.
A unique Class 2 transposable element was discovered as a 128 base pair insertion in an exon of the maize waxy coding region in the wxB2 mutant allele. Database searches identified related elements (on average about 70% sequence identity) in the introns or the 5′ or 3′ flanking sequences of many maize coding regions. This new family was called Tourist. Almost one third of all sequenced maize coding regions contain a Tourist element as do the coding regions of other members of the grass tribe including rice, sorghum and barley. An insertion into a Tourist element led to the discovery of another element family called Stowaway, in the coding regions of both monocotyledonous and dicotyledonous plants. Finally, a systematic search of all available rice genomic sequences identified the new element families Gaijin, Castaway, Ditto, Wanderer, Explorer and revealed that short inverted-repeat elements were the predominant repeat sequence associated with rice coding regions (Bureau et al. (1996)
Proc. Natl. Acad. Sci. USA,
93 8524-8529). These elements formed a unique collection of inverted-repeat transposons referred to as miniature inverted-repeat transposable elements (MITEs).
MITEs have been identified in all flowering plants that have significant genomic nucleotide sequences present in databases. For instance, MITE families have been found in maize, rice (including Gaijin, Castaway, Ditto, Wanderer, Explorer; Snap, Crackle, and Pop), bell pepper (Alien), and alfalfa (Bigfoot). The first MITE family from Arabidopsis, Emigrant, was recently described. Most characterized MITE families in plants appear to be relatively ancient components of genomes since family members were only distantly related to each other (70% sequence identity on average) and insertion sites were usually not polymorphic among members of the same species (Bureau et al. (1992)
The Plant Cell
4, 1283-1294; Bureau et al. (1994)
Proc. Natl. Acad. Sci. USA,
91, 1411-1415; Bureau et al. (1994)
The Plant Cell,
6, 907-916).
MITEs are not restricted to flowering plants. MITE families have been described in insects (
Aedes aegypti,
the yellow fever mosquito),
C. elegans,
and even humans (trigger 1 and 2).
Despite the prevalence of MITEs in plant genomes little is known about their biology including, for instance, their distribution. This largely reflects the fact that most MITEs have been identified through database searches (see, for instance, Bureau et al. (1992)
The Plant Cell,
4, 1283-1294; Bureau et al. (1994)
Proc. Natl. Acad. Sci. USA,
91, 1411-1415; Bureau et al. (1994)
The Plant Cell,
6, 907-916. For this reason, much of what is known about this important class of elements is restricted to MITE identification, categorization and descriptions of their presence in genic regions. It is not currently known, for example, whether their association with coding regions reflects a true target site preference or whether this is merely an artifact of identifying elements by searching the gene-rich databases. Recently, it was shown that in a 225 kilobase region of the maize genome, putative MITEs were found within genic regions, and not in nongenic regions (Tikhonov et al. (1999)
Proc. Natl. Acad. Sci. USA,
96, 7409-7414) However, 225 kilobases represents less than 0.0001% of total maize DNA, thus it is unclear if these results can be extrapolated to the entire maize genome.
The investigation of genome structure has been accelerated by the use of in vitro methods that detect variation in the DNA sequence in the genomes between members of a species or closely related species. This variation at different locations in the genome is unique for each individual member of a species. These in vitro methods detect the variation, and produce what is referred to as a DNA fingerprint for an individual. Typically, the more closely related two individuals, the more similar the DNA fingerprint from each individual. DNA sequence differences detected by DNA fingerprinting, including single base pair changes as well as large deletions or additions, are referred to as polymorphisms. A polymorphism provides a marker for a specific location on a chromosome in the individual containing the polymorphism. A marker is typically detected as a DNA fragment.
Since the advent of these in vitro methods in the early 1980s, numerous methods for detecting polymorphisms that mark chromosomes have been developed. For instance, restriction fragment length polymorphism (RFLP), DNA amplification fingerprinting, cleaved amplified polymorphisms, randomly amplified polymorphic DNA, arbitrary primed-polymerase chain reaction, random amplified microsatellite polymorphism, simple sequence repeat, amplified fragment length polymorphism (AFLP) (Zabeau, EP Pat. No. 0 534 858 A1) and sequence-specific amplification polymorphisms (Waugh et al. (1997)
Mol. Gen. Genet.,
253, 687-694) have made their way into use in plant breeding and genetics. In general, the markers that are produced by each of these methods are randomly distributed throughout the genome and allow saturated genome coverage if enough markers are developed.
Typically, genomes contain nongenic regions, i.e., regions that do not contain coding regions. This is particularly true of plants where up to 99.5% of the genome can be nongenic. Nongenic regions are made up of mainly repetitive DNA, i.e., regions of DNA having nucleotides sequences that are present multiple times in the genome. Interspersed in nongenic are regions containing coding regions. These regions are referred to as genic regions and are made up of low or single copy regions of DNA. Typically, a large fraction of the markers generated by in vitro methods that detect variation in the DNA sequence are located in nongenic regions. Consequently, there is an increased cost in generating and mapping excessive numbers of markers.
SUMMARY OF THE INVENTION
The large plant genomes generally contain genes interspersed with much longer blocks of repetitive DNA. Given this organization, it would be highly desirable to have polymorphic markers that are located preferentially in genic regions. It would be even more desirable if these markers were present in high numbers in the genic regions. The present invention discloses that miniature inverted repeat transposable elements (MITEs) are polymorphic markers located preferentially in genic regions. The invention presents the first analysis of the distribution of MITEs that includes an entire genome, i.e., the analysis is not confined to genic regions or to a limited portion of a genome. This analysis indicates that MITEs are preferentially located in genic regions. This analysis of MITEs also unexpectedly showed that MITEs are polymorphic. The polymorphic nature of MITEs was surprising because other transposable elements associated with genic regions, for instance Alu elements in humans, are usually in the same position in all individuals of a species. The polymorphism of MITEs, coupled with their genic preference, indicates that they are a major factor in generating allelic diversity.
An advantage of the methods of the present invention over other in vitro methods that detect DNA variation is that since markers generated using MITEs are preferentially located in genic regions, less markers must be generated and mapped. Thus, costs a
Casa Alexandra M.
Wessler Susan R.
Horlick Kenneth R.
Mueting Raasch & Gebhardt, P.A.
Spiegler Alexander H.
The University of Georgia Research Foundation Inc.
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