Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-04-01
2001-05-15
Horlick, Kenneth R. (Department: 1600)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000
Reexamination Certificate
active
06232061
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the manipulation of genetic materials and, more specifically, to the methods for cloning a DNA sequence of interest.
BACKGROUND OF THE INVENTION
Success in identifying a homologue of a prototype gene rests on several factors. Foremost is the rate of sequence divergence, which will determine whether sufficient remnants of a primordial sequence are retained over time to enable cloning and identification of homologous genes over great evolutionary distances.
For example, if sequence similarity between the prototype gene in one organism and its homologue in another organism (which is to be cloned) is relatively high, extending across much of the molecule, then one expects any part of the prototype sequence to provide an adequate probe to detect the homologue.
Further, even if sequence similarity is not high, one may still be able to clone the homologue by subdividing the prototype gene into smaller fragments and test each for its binding to DNA of the target organism. However, only fragments that harbor conserved DNA sequence elements are expected to detect the homologue. An alternative approach to the development of probes depends on the design of degenerate oligonucleotides. This approach requires a consensus protein sequence derived from analysis of multiple homologues of the desired gene. Such analysis appears essential for successful long distance homology cloning.
Long distance homology cloning pertains to periods of time in excess of 600-700 million years. Though the method described in this invention illustrates how to clone across phyla of metazoan organisms, it can be applied to longer time frames or to crossing phyla within other kingdoms, such as plants or fungi. A useful point of reference pertinent to metazoans is when the coelomic lineage, which led to both protostomes and deuterostomes, separated from pseudocoelomates and acoelomic metazoans.
The present invention addresses the central question in long distance homology cloning; namely, what steps are necessary in order to develop probes capable of discriminating homologues from non-homologous DNA?
SUMMARY OF THE INVENTION
In general, the invention, in one aspect, features a method for isolating a structural homologue of a first, higher organism which is structurally homologous with a gene of a second, lower organism, the method comprising the steps of:
a. using two or more oligonucleotides based on two or more regions of the gene to detect hybridization signals or candidate homologues in the genome of a third organism which is positioned phylogenetically between the first organism and the second organism;
b. sequencing the hybridization signals or candidate homologues;
c. selecting structural homologues of the gene based on multiple resemblance in structural characteristics;
d. using additional two or more oligonucleotides based on two or more conserved regions among the structural homologues and the gene to detect hybridization signals in the genome of an additional organism which is phylogenetically positioned between the first organism and the third organism; and
e. repeating steps b through d until the gene of the first organism is isolated.
In the above method, the oligonucleotides are either hybridizing probes for detection of hybridization signals or are PCR primers for detection of candidate homologues. When hybridizing probes are used, it is preferred that candidate homologues be selected in step b based on sameness in arrangement or strandedness or both of the probe-binding regions before sequencing the hybridization signals. The term “sameness” here and below refers to a comparison between a probe- or primer-binding region and its corresponding probe or primer. Similarly, the term “resemblance” as recited in step c and in other places below refers to a comparison between the structural homologue and the gene. Note that the length of the oligonucleotides herein (either probes or primers) ranges from 15 nucleotides to the full length of the gene or its structural homologues (either cDNAs or genomic sequences).
Further, either primers or probes are used, it is preferred that in step b the probe- or primer-binding regions or their surrounding regions be first sequenced and putative homologues be selected based on presence of an open reading frame in the sequenced probe- or primer-binding regions alone or together with the sequenced surrounding regions. Other criteria for selecting putative homologues include presence of a splice site(s) in the vicinity of the probe- or primer-binding regions, and sequence similarity between the probe- or primer-binding regions and the probes or primers. Clearly, one can also select a putative homologue further based on sequence similarity beyond the probe- or primer-binding regions. [The term “similarity” here and below again refers to a comparison between the surrounding regions of the probe- or primer-binding sites and their counterparts in the gene.] Also, it is desirable that only the hybridization signals that are detected by at least two of the two or more oligonucleotides are sequenced. Alternatively, one can prioritize the hybridization signals to be sequenced by first examining the presence of dispersed sequence similarity with the oligonucleotides based on restriction/hybridization analysis. See Example 3 below for a detailed description of this analysis.
In the above-described method, the oligonucleotides are used to screen a DNA library, e.g., a genomic library or a cDNA library. Further, the oligonucleotides can also be applied to genomic DNA analyzed on a Southern blot in a manner well known in the art. Also see working examples set forth below. Note that one can also use the oligonucleotides as a primer pair in a PCR amplification with RNA (e.g., total RNA or mRNA), reverse transcribed RNA (i.e., cDNA), or DNA (e.g., ds-cDNA or genomic DNA which is cloned/fractionated or otherwise) being the template.
The oligonucleotides used in this method are preferably degenerate. Further, the oligonucleotides used in step e can be based on intra-species conserved regions among members in a gene family.
The terms “lower organism” and “higher organisms” used herein are referred to according to the following scheme. Lower organisms are represented by the prototype organism, that is the organism from which the first gene sequence of interest is isolated and characterized. Organisms are considered progressively higher as one proceeds towards the human lineage, with human considered the highest organism. For a detailed discussion of the organisms selected based on their phylogenetic relationships, see Selection of Organisms below in “Description of the Preferred Embodiments”.
In this method, selection of the third organism, the additional organism, or both can be based on the rate of change for a protein during evolution (e.g., see FIG.
2
). Note that both in this method and in the methods described below the additional organism is also the first organism at the last cloning cycle. The term “conserved regions” recited in step d of this method (as well as mentioned below) refer to the conservation of peptide residues among the organisms during evolution.
The first organism, can be a deuterostome, a protostome, or a vertebrate (such as a mammal, which can be human). The second organism, on the other hand, can be a nematode, such as
Caenorhabditis elegans, Caenorhabditis remanei, Caenorhabditis briggsae, Panagrellus redivivus, Ascaris suum, Bruggia malayi, Haemonchus contortus,
or
Rhabditis maupasi.
The gene can be a cell death gene, such as ced-1, ced-2, ced-3, ced-4, ced-5, ced-6, ced-7, ced-8, ced-9, ced-10, mec-4, mec-6, deg-1, deg-3, egl-1, nuc-1, lin-24, or lin-33. For a review of the above-listed cell death genes, see Horvitz et al. In
New Biological Approaches to Neurological Disorders: Pathogenesis and Treatment,
Dahlem Konferenzen, Berlin, Aug. 5-10, 1990.
The invention, in another aspect, features a method for identifying a structural homologue in a first organism which is structurally h
Johnson Carl D.
Marchionni Mark Andrew
Cambridge NeuroScience, Inc.
Corless Peter F.
Edwards & Angell LLP
Horlick Kenneth R.
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