# Patent

## Title
C. elegans gro-1 gene

## Abstract

The invention relates to the identification of gro-1 gene and to demonstrate that the gro-1 gene is involved in the control of a central physiological clock. Also disclosed are four other genes located within the same operon as the gro-1 gene.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of PCT/CA98/00803 filed Aug. 20, 1998, now at the national phase, and claiming priority on Canadian patent application serial number 2,210,251 filed Aug. 25, 1997, now abandoned.


BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to the identification of gro-1 gene and four other genes located within the same operon and to show that the gro-1 gene is involved in the control of a central physiological clock.
(b) Description of Prior Art
The gro-1 gene was originally defined by a spontaneous mutation isolated from of a Caenorhabditis elegans strain that had recently been established from a wild isolate (J. Hodgkin and T. Doniach, Genetics 146: 149-164 (1997)). We have shown that the activity of the gro-1 gene controls how fast the worms live and how soon they die. The time taken to progress through embryonic and post-embryonic development, as well as the life span of gro-1 mutants is increased (Lakowski and Hekimi, Science 272: 1010-1013, (1996)). Furthermore, these defects are maternally rescuable: when homozygous mutants (gro-1/gro-1) derive from a heterozygous mother (gro-1/+), these animals appear to be phenotypically wild-type. The defects are seen only when homozygous mutants derive from a homozygous mother (Lakowski and Hekimi, Science 272: 10101013, (1996)). In general, the properties of the gro-1 gene are similar to those of three other genes, clk-1, clk-2 and clk-3 (Wong et al., Genetics 139: 1247-1259 (1995); Hekimi et al., Genetics, 141: 1351-1367 (1995); Lakowski and Hekimi, Science 272: 1010-1013, (1996)), and this combination of phenotypes has been called the Clk (“clock”) phenotype. All four of these genes interact to determine developmental rate and longevity in the nematode. Detailed examination of the clk-1 mutant phenotype has led to the suggestion that there exists a central physiological clock which coordinates all or many aspects of cellular physiology, from cell division and growth to aging. All four genes have a similar phenotype and thus appear to impinge on this physiological clock.
It would be highly desirable to be provided with the molecular identity of the gro-1 gene.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide the molecular identity of the gro-1 gene and four other genes located within the same operon.
In accordance with the present invention there is provided a gro-1 gene which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein gro-1 is located within an operon and gro-1 mutants have a longer life and a altered cellular metabolism relative to the wild-type.
In accordance with a preferred embodiment, the gro-1 gene of the present invention codes for a GRO-1 protein having the amino acid sequence set forth in FIGS. 3A-3B (SEQ ID. NO:2).
The gro-1 gene is located within an operon which has the nucleotide sequence set forth in SEQ. ID NO:1 and which also codes for four other genes, referred as gop-1, gop-2, gop-3 and hap-1 genes.
In accordance with a preferred embodiment, the gop-1 gene of the present invention codes for a GOP-1 protein having the amino acid sequence set forth in FIGS. 13A-13C (SEQ ID. NO:4).
In accordance with a preferred embodiment, the gop-2 gene of the present invention nodes for a GOP-2 protein having the amino acid sequence set forth in FIG. 14 (SEQ ID. NO:5).
In accordance with a preferred embodiment, the gop-3 gene of the present invention codes for a GOP-3 protein having the amino acid sequence set forth in FIGS. 15A-15B (SEQ ID. NO:6).
In accordance with a preferred embodiment, the hap-1 gene of the present invention codes for a HAP-1 protein having the amino acid sequence set forth in FIG. 16 (SEQ ID. NO:7).
In accordance with a preferred embodiment of the present invention, the gro-1 gene is of human origin and has the nucleotide sequence set forth in FIG. 8 (SEQ ID. NO:3).
In accordance with a preferred embodiment of the present invention, there is provided a mutant GRO-1 protein which has the amino acid sequence set forth in FIG. 3C.
In accordance with the present invention there is also provided a GRO-1 protein which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein said GRO-1 protein is encoded by the gro-1 gene identified above.
In accordance with a preferred embodiment of the present invention, there is provided a GRO-1 protein which has the amino acid sequence set forth in FIGS. 3A-3B (SEQ ID. NO:2).
In accordance with a preferred embodiment of the present invention, there is provided a GOP-1 protein which has the amino acid sequence set forth in FIGS. 13A-13C (SEQ ID. NO:4).
In accordance with a preferred embodiment of the present invention, there is provided a GOP-2 protein which has the amino acid sequence set forth in FIG. 14 (SEQ ID. NO:5).
In accordance with a preferred embodiment of the present invention, there is provided a GOP-3 protein which has the amino acid sequence set forth in FIGS. 15A-15B (SEQ ID. NO:6).
In accordance with a preferred embodiment of the present invention, there is provided a HAP-1 protein which has the amino acid sequence set forth in FIG. 16 (SEQ ID. NO:7).
In accordance with the present invention there is also provided a method for the diagnosis and/or prognosis of cancer in a patient, which comprises the steps of:

    a) obtaining a tissue sample from said patient;
    b) analyzing DNA of the obtained tissue sample of step a) to determine if the human gro-1 gene is altered; wherein alteration of the human gro-1 gene is indicative of cancer.


In accordance with the present invention there is also provided a mouse model of aging and cancer, which comprises a gene knock-out of murine gene homologous to gro-1.
In accordance with the present invention there is provided the use of compounds interfering with enzymatic activity of GRO-1, GOP-1, GOP-2, GOP-3 or HAP-1 for enhancing longevity of a host.
In accordance with the present invention there is provided the use of compounds interfering with enzymatic activity of GRO-1, GOP-1, GOP-2, GOP-3 or HAP-1 for inhibiting tumorous growth.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates the genetic mapping of gro-1;
FIG. 1B illustrates the physical map of the gro-1 region;
FIG. 2A illustrates cosmid clones able to rescue the gro-1 (e2400) mutant phenotype;
FIG. 2B illustrates the genes predicted by Genefinder, the relevant restriction sites and the fragments used to subclone the region;
FIGS. 3A-3C illustrate the genomic sequence and translation of the C. elegans gro-1 gene (SEQ ID NO: 2 and 66);
FIG. 3D illustrates the predicted mutant protein (SEQ ID NO: 64 and 65);
FIG. 4A illustrates the five genes of the gro-1 operon (SEQ. ID. NO:1);
FIG. 4B illustrates the transplicing pattern of the five genes of the gro-1 operon;
FIGS. 5A-5B illustrates the alignment of gro-1 with the published sequences of the E. coli (P16384) and yeast (PO7884) enzymes;
FIG. 6 illustrates the biosynthetic step catalyzed by DMAPP transferase (MiaAp in E. coli, Mod5p in S. cerevisiae, and GRO-1 in C. elegans);
FIG. 7 illustrates the alignment of the predicted HAP-1 amino acid sequence with homolgues from other species (SEQ ID NO: 69, 7, 70 and 71);
FIG. 8 illustrates the full mRNA sequence of human homologue of gro-1 referred to as hgro-1 (SEQ. ID. NO:3);
FIGS. 9A-9B illustrates a comparison of the conceptual amino acid sequences for GRO-1 (SEQ ID NO: 2) and hgro-1p(SEQ ID NO: 63);
FIG. 10 illustrates a conceptual translation of a partial sequence of the Drosophila homologue of gx-o-1 (AA816785) (SEQ ID NO: 72);
FIG. 11 illustrates the structure of pMQB (SEQ ID NO: 73 and 2);
FIG. 12 illustrates construction of pMQ18;
FIGS. 13A-13C illustrate the genomic sequence and translation of the gop-1 gene (SEQ ID NO: 73 and 4);
FIG. 14 illustrates the genomic sequence and translation of the gop-2 gene (SEQ ID NO: 74 and 75);
FIGS. 15A-15B illustrate the genomic sequence and translation of the gop-3 gene (SEQ ID NO: 75 and 6); and
FIG. 16 illustrates the genomic sequence and translation of the hap-1 gene (SEQ ID NO: 6 and 77).



DETAILED DESCRIPTION OF THE INVENTION
The Gro-1 Phenotype
In addition to the previously documented phenotypes, we recently found that gro-1 mutants were temperature-sensitive for fertility. At 25° the progeny of these mutants is reduced so much that a viable strain cannot be propagated. In contrast, gro-1 strains can easily be propagated at 15 and 20° C.
We also discovered that the gro-1 (e2400) mutation increases the incidence of spontaneous mutations. As gro-1(e2400) was originally identified in a nonstandard background (Hodgkin and Doniach, Genetics. 146: 149-164 (1997)), we first backcrossed the mutations 8 times against N2, the standard wild type strain. We then undertook to examine the gro-1 strain and N2 for the occurrence of spontaneous mutants which could be identified visually. We focused on the two class of mutants which are detected the most easily by simple visual inspection, uncoordinated mutants (Unc) and dumpy mutants (Dpy). We examined 8200 wild type worms and found no spontaneous visible mutant. By contrast, we found 6 spontaneous mutants among 12500 gro-1 mutants examined. All mutants produced entirely mutant progeny indicating that they were homozygous.













Name
Orientation
Sequence (5′-3′)
SEQ ID NO:







SHP91
forward
CGAACACTTTATATTTCTCG
SEQ. ID. NO:8 


SHP92
reverse
GATAGTTCCCTTCGTTCGGG
SEQ. ID. NO:9 


SHP93
forward
TTTCTGGATTTTAACCTTCC
SEQ. ID. NO:10


SHP94
forward
TTTCCGAGAAGTCACGTTGG
SEQ. ID. NO:11


SHP95
reverse
TACAGGAATTTTTGAACGGG
SEQ. ID. NO:12


SHP96
forward
CTTCAGATGACGTGGATTCC
SEQ. ID. NO:13


SHP97
forward
GGAATCCGAAAAAGTGAACT
SEQ. ID. NO:14


SHP98
forward
AAGAGATACACTCAATGGGG
SEQ. ID. NO:15


SHP99
reverse
ATCGATACCACCGTCTCTGG
SEQ. ID. NO:16


SHP109
reverse
TTGAATCTACACTAATCACC
SEQ. ID. NO:17


SHP100
reverse
CCAATTATCTTTTCCAGTCA
SEQ. ID. NO:18


SHP110
forward
ACATTATAAAGTTACTGTCC
SEQ. ID. NO:19


SHP118
forward
TTTTAGTTAAAGCATTGACC
SEQ. ID. NO:20


SHP119
reverse
ACATCTTTATCCATTTCTCC
SEQ. ID. NO:21


SHP120
forward
TGCAAAGGCTCTGGAACTCC
SEQ. ID. NO:22


SHP129
reverse
AAAAACCACTTGATATAAGG
SEQ. ID. NO:23


SHP130
reverse
CATCCAAAAGCAGTATCACC
SEQ. ID. NO:24


SHP134
forward
TTAATTGGATGCAAGCACCCC
SEQ. ID. NO:25


SHP135
reverse
ATTACTATACGAACATTTCC
SEQ. ID. NO:26


SHP138
forward
TTGTAAAGGCGTTAGTTTGG
SEQ. ID. NO:27


SHP139
forward
CAGGAGTATTTGGTGATGCG
SEQ. ID. NO:28


SHP140
forward
CGACGGGGAGAAGGTGACGG
SEQ. ID. NO:29


SHP141
reverse
AAAACTTCTACCAACAATGG
SEQ. ID. NO:30


SHP142
reverse
CGTAATCTCTCTCGATTAGC
SEQ. ID. NO:31


SHP143
reverse
CCGTGGGATGGCTACTTGCC
SEQ. ID. NO:32


SHP144
reverse
TGGATTTGTGGCACGAGCGG
SEQ. ID. NO:33


SHP145
reverse
TTGATTGCCTCTCCTCGTCC
SEQ. ID. NO:34


SHP146
reverse
ATCAACATCTGATTGATTCC
SEQ. ID. NO:35


SHP151
forward
CAGCGAGCGCATGCAACTATATATTGA
SEQ. ID. NO:36




GCAGG


SHP159
forward
AATAAATATTTAAATATTCAGATATACC
SEQ. ID. NO:37




CTGAACTCTACAG


SHP160
reverse
AAACTGTAGAGTTCAGGGTATATCTGA
SEQ. ID. NO:38




ATATTTAAATATTTATTC


SHP161
forward
GTACGTGGAGCTCTGCAACTATATATT
SEQ. ID. NO:39




GAGCAGG


SHP162
reverse
ATGACACTGCAGGATAGTTCCCTTCGT
SEQ. ID. NO:40




TCGGG


SHP163
forward
GTGTTGCATCAGTTCATTCC
SEQ. ID. NO:41


SHP164
forward
GCTGTGCTAGAAGTCAGAGG
SEQ. ID. NO:42


SHP165
reverse
GTTCTCCTTGGAATTCATCC
SEQ. ID. NO:43


SHP170
reverse
AGTATATCTAGATGTGCGAGTCTCTGC
SEQ. ID. NO:44




CAATT


SHP171
reverse
AGTAATTGTACATTTAGTGG
SEQ. ID. NO:45


SHP172
forward
ATTAACCTTACTTACTTACC
SEQ. ID. NO:46


SHP173
forward
CTAAACTAAGTAATATAACC
SEQ. ID. NO:47


SHP174
reverse
GTTGATTCTTTGAGCACTGG
SEQ. ID. NO:48


SHP175
forward
AATTCGACCAATTACATTGG
SEQ. ID. NO:49


SHP176
reverse
AACATAGTTGTTGAGGAAGG
SEQ. ID. NO:50


SHP177
forward
AATTAATGGAGATTCTACGG
SEQ. ID. NO:51


SHP178
forward
TCAGCATCTAGAAATGCAGG
SEQ. ID. NO:52


SHP179
reverse
CGAATGTCAACATTCACTGG
SEQ. ID. NO:53


SHP180
forward
CTTAACCTGATGTGTACTCG
SEQ. ID. NO:54


SHP181
forward
ATGAAGCTTTAGAGGATGCC
SEQ. ID. NO:55


SHP182
forward
CGACGAATTTCTGGAGTCGG
SEQ. ID. NO:56


SHP183
reverse
ACTGCATTATCCATTAATCC
SEQ. ID. NO:57


SHP184
reverse
CACCCAAATAACATCTATCC
SEQ. ID. NO:58


SHP185
forward
TTTAACCTCATCTTCGCTGG
SEQ. ID. NO:59


SHP190
forward
ATGTTCCGCAAGCTTGGTTC
SEQ. ID. NO:60


SL1
forward
TTTAATTACCCAAGTTTGAG
SEQ. ID. NO:61


SL2
forward
TTTTAACCCAGTTACTCAAG
SEQ. ID. NO:62









Positional Cloning of Gro-1

gro-1 lies on linkage group III, very close to the gene clk-1. To genetically order gro-1 with respect to clk-1 on the genetic map, 54 recombinants in the dpy-17 to lon-1 interval were selected from among the self progeny of a strain which was unc-79(e1030)++clk-1(e2519) lon-1(e678)+/+dpy-17(e164) gro-1(e2400)+sma-4,(e729). Three of these showed neither the Gro-1 nor the Clk-1 phenotypes, but carried unc-79 and sma-4, indicating that these recombination events had occurred between gro-1 and clk-1. From the disposition of the markers, this showed that the gene order was dpy-17 gro-1 clk-1 lon-1, and the frequency of events indicated that the gro-1 to clk-1 distance was 0.03 map units. In this region of the genome, this corresponds to a physical map distance of  ̃20 kb.
Several cosmids containing wild-type DNA spanning this region of the genome were tested by microinjection into gro-1 mutants for their ability to complement the gro-1(e2400) mutation (FIG. 1). gro-1 was mapped between dpy-17 and lon-1 on the third chromosome, 0.03 m.u. to the left of clk-1 (Fig. A).
Based on the above genetic mapping, gro-1 was estimated to be approximately 20 kb to the left of clk-1. Eight cosmids (represented by medium bold lines) were selected as candidates for transformation rescue (FIG. 1B). Those which were capable of rescuing the gro-1(e2400) mutant phenotype are represented as heavy bold lines (FIG. 1B).
Of these, only B0498, C34E10 and ZC395 were able to rescue the mutant phenotype. Transgenic animals were fully rescued for developmental speed. In addition, the transgenic DNA was able to recapitulate the maternal rescue seen with the wild-type gene, that is, mutants not carrying the transgenic DNA but derived from transgenic mothers display a wild type phenotype. The 7 kb region common to the three rescuing cosmids had been completely sequenced, and this sequence was publicly available.
We generated sabclones of ZC395 and assayed them for rescue (FIG. 2). The common 6.5 kb region is blown up in part B. B0498 has not been sequenced and therefore its ends can not be positioned and are therefore represented by arrows.
One subclone pMQ2, spanned 3.9 kb and was also able to completely rescue the growth rate defect and recapitulate the maternal effect. The sequences in pMQ2 potentially encodes two genes. However, a second subclone, pMQ3, which contained only the first of the potential genes (named ZC395.7 in FIG. 2A), was unable to rescue.
Furthermore., frameshifts which would disrupt each of the two genes' coding sequences were constructed in pMQ2 and tested for rescue. Disruption of the first gene (in pMQ4) did not eliminate rescuing ability, but disruption of the second gene (in pMQ5) did. This indicates that the gro-1 rescuing activity is provided by the second predicted gene.
pMQ2 was generated by deleting a 29.9 kb SpeI fragment from ZC395, leaving the left-most 3.9 kb region containing the predicted genes ZC395.7 and ZC395.6 (FIG. 2B). pMQ3 was created in the same fashion, by deleting a 31.4 kb NdeI fragment from ZC395, leaving only ZC395.7 intact. In pMQ4, a frameshift was induced in ZC395.7 by degrading the 4 bp overhang of the ApaI site. A frameshift was also induced in pMQ5 by filling in the 2 bp overhang of the NdeI site found in the second exon of ZC395.6. These frameshifts presumably abolish any function of ZC395.7 and ZC395.6 respectively. The dotted lines represent the extent of frameshift that resulted from these alterations.
To establish the splicing pattern of this gene, cDNAs encompassing the 5′ and 3′ halves of the gene were produced by reverse transcription-PCR and sequenced (FIG. 3).
This revealed that the gene is composed of 9 exons, spans  ̃2 kb, and produces an mRNA of 1.3 kb. To confirm that this is indeed the gro-1 gene, genomic DNA was amplified by PCR from a strain containing the gro-1(e2400) mutation and the amplified product was sequenced. A lesion was found in the 5th exon, where a 9 base-pair sequence has been replaced by a 2 base-pair insertion, leading to a frameshift (FIG. 3C). FIG. 3C illustrates those residues which differ from wild type are in bold.
The reading frame continues out-of-frame for another 33 residues before terminating.
FIGS. 3A-B illustrate the coding sequence in capital letters, while the introns, and the untranslated and intergenic sequence are in lower case letters. The protein sequence is shown underneath the coding sequence. Position 1 of the nucleotide sequence is the first base after the SL2 trans-splice acceptor sequence. Position 1 of the protein sequence is the initiator methionine. All PCR primers used for genomic and cDNA amplification are represented by arrows. For primers extending downstream (arrows pointing right) the primer sequence corresponds exactly to, the nucleotides over which the arrow extends. But for primers extending upstream (arrows pointing left) the primer sequence is actually the complement of the sequence under the arrow. In both cases the arrow head is at the 3′ end of the primer. The sequence of the two primers which flank gro-1 (SHP93 and SHP92) are not represented in this figure. Their sequences are: SHP93 TTTCTGGATTTTAACCTTCC (SEQ. ID. NO:10) and SHP92 GATAGTTCCCTTCGTTCGGG (SEQ. ID. NO:9). The wild type splicing pattern was determined by sequencing of the cDNA. Identification of the e2400 lesion was accomplished by sequencing the e2400 allele. The e2400 lesion consists of a 9 bp deletion and a 2 bp insertion at position 1196, resulting in a frameshift.
Gro-1 is Part of a Complex Operon (FIGS. 3A-3B)
Amplification of the 5′ end of gro-1 from cDNA occurred only when the trans-spliced leader SL2 was used as the 5′ primer, and not when SL1 was used. SL2 is used for trans-splicing to the downstream gene when two genes are organized into an operon (Spieth et al., Cell 73: 521-532 (1993); Zorio et al., Nature 372: 270-272 (1994)). This indicates that at least one gene upstream of gro-1 is co-transcribed with gro-1 from a common promoter. We found that sequences from the 5′ end of the three next predicted genes upstream of gro-1 (ZC395.7, C34E10.1, and C34E10.2) all could only be amplified with SL2. Sequences from the fourth predicted upstream gene (C34E10.3), however, could be amplified with neither spliced leader, suggesting that it is not trans-spliced. The distance between genes in operons appear to have an upper limit (Spieth et al., Cell 73: 521-532 (1993); Zorio et al., Nature 372: 270-272 (1994)), and no gene is predicted to be close enough upstream of C34E10.3 or downstream of gro-1 to be co-transcribed with these genes. Our findings suggest therefore that gro-1 is the last gene in an operon of five co-transcribed genes (FIG. 4).
Nested PCR was used to amplify the 5 end of each gene. SL1 or SL2 specific primers were used in conjunction with a pair of gene-specific primers cDNA generated by RT-PCR using mixed stage N2 RNA was used as template in the nested PCR. FIG. 4A illustrates a schematic of the gro-1 operon showing the coding sequences of each gene and the primers (represented by flags) used to establish the trans-splicing patterns.
FIG. 4B illustrates the products of the PCR with SL1 and SL2 specific primers for each of the five genes. The sequences of the primers used are as follows: SL1: TTTAATTACCCAAGTTTGAG (SEQ. ID. NO:61), SL2: TTTTAACCCAGTTACTCAAG (SEQ. ID. NO:62), SHP141: AAAACTTCTACCAACAATGG (SEQ. ID. NO:30), SHP142: CGTAATCTCTCTCGATTAGC (SEQ. ID. NO:31), SHP143: CCGTGGGATGGCTACTTGCC (SEQ. ID. NO:32), SHP144: TGGATTTGTGGCACGAGCGG (SEQ. ID. NO:33), SHP145: TTGATTGCCTCTCCTCGTCC (SEQ. ID. NO:34), SHP146: ATCAACATCTGATTGATTCC (SEQ. ID. NO:35), SHP130: CATCCAAAAGCAGTATCACC (SEQ. ID. NO:24); SHP119: ACATCTTTATCCATTTCTCC (SEQ. ID. NO:21), SHP95: TACAGGAATTTTTGAACGGG (SEQ. ID. NO:12), SHP99: ATCGATACCACCGTCTCTGG (SEQ. ID. NO:16).
The gene immediately upstream of gro-1, has homology to the yeast gene HAM1, and we have renamed the gene hap-1. We have established its splicing pattern by reverse transcription PCR and sequencing. This revealed that hap-1 is composed of 5 exons and produces an mRNA of 0.9 kb. We also found that sequences which were predicted to belong to ZC395.7 (now hap-1) are in fact spliced to the exons of C34E10.1. This is consistent with our finding that hap-1 is SL2 spliced as it puts the end of the C34E10.1 very close to the start of hap-1 (FIG. 4).
The Gro-1 Gene Product
Conceptual translation of the gro-1 transcript indicated that it encodes a protein of 430 amino acids highly similar to a strongly conserved cellular enzyme: dimethylallyldiphosphate:tRNA dimethylallyltransferase (DMAPP transferase). FIG. 5 shows an alignment of gro-1 with the published sequences of the E. coli (P16384) and yeast (P07884) enzymes. Residues where the biochemical character of the amino acids is conserved are shown in bold. Identical amino acids are indicated further with a dot. The ATP/GTP binding site and the 0.30 C2H2 zinc finger site are predicted and hot experimental. The point at which the gro-1(e2400) mutation alters the reading frame of the sequence is shown. The two alternative initiatior methionines in the yeast sequence, and the putative corresponding methionines in the worm sequence, are underlined.
Database searches also identified a homologous human expressed sequence tag (Genbank ID: Z40724). The human clone has been used to derive a sequence tagged site (STS). This means that the genetic and physical position of the human gro-1 homologue is known. It maps to chromosome 1, 122.8 cR from the top of Chr 1 linkage group and between the markers D1S255 and D1S2861. This information was found in the UniGene database or the National Center for Biotechnology Information (NCBI). We have sequenced Z40724 by classical methods but found that Z40724 is not a full length cDNA clone as it does not contain an initiator methionine nor the poly A tail. We used the sequence of Z40724 to identify further clones by database searches. We found one clone (Genbank ID: AA332152) which extended the sequence 5′ by 28 nucleotides, as well as one clone (Genebank ID: AA121465) which extended the sequence substantially in the 3′ direction but didn't include the poly A tail. We then used AA121465 to identify an additional clone (AA847885) extending the sequence to the poly A tail. FIG. 8 shows the full sequence with the putative initiator ATG shown in bold and the sequence of Z60724 is shown underlined. A comparison of the conceptual amino acid sequences for GRO-1 (SEQ ID NO:2) and hgro-1p as deduced from SEQ ID NO:3, is shown in FIG. 9. Amino acid identities are indicated by a dot. Both sequences contain a region with a zinc finger motif which is shown underlined.
An additional metazoan homologue is represented by Drosophila EST: Genbank accession: AA816785. In E. colii and other bacteria, the gene encoding DMAPP transferase is called miaA (a.k.a trpX) and is called modS in yeast. DMAPP transferase catalyzes the modification of adenosine 37 of tRNAs whose anticodon begins with U (FIG. 6).
In these organisms the enzyme has been shown to use dimethylallyldiphosphate as a donor to generate dimethylallyl-adenosine (dma6A37), one base 3′ to the anticodon (for review and biochemical characterization of the bacterial enzyme see Persson et al., Biochimie 76: 1152-1160 (1994); Leung et al., J Biol Chem 272: 13073-13083 (1997); Moore and Poulter, Biochemistry 36: 604-614 (1997)). In earlier literature this modification is often referred to as isopentenyl adenosine (i6A37).
The high degree of conservation of the protein sequence between GRO-1 and DMAPP in S. cerevisiae and E. coli suggest that GRO-1 possesses the same enzymatic activity as the previously characterized genes. The sequence contains a number of conserved structural motifs (FIG. 5), including a region with an ATP/GTP binding motif which is generally referred to as the ‘A’ consensus sequence (Walker et al., EMBO J 1: 945-951 (1982)) or the ‘P-loop’ (Saraste et al., Trends Biochem Sci 15: 430-434 (1990)).
In addition, at the C-terminal end of the GRO-1 sequence, there is a C2H2 zinc finger motif as defined by the PROSITE database. This type of DNA-binding motif is believed to bind nucleic acids (Klug and Rhodes, Trends Biochem Sci 12: 464-469 (1987)). Although there appears to be some conservation between the worm and yeast sequences in the C-terminus end of the protein (FIG. 5), including in the region encompassing the zinc finger in GRO-1, the zinc finger motif per se is not conserved, in yeast but is present in humans (FIG. 9).
In yeast DMAPP transferase is the product of the MOD5 gene, and exists in two forms: one form which is targeted principally to the mitochondria, and one form which is found in the cytoplasm and nucleus. These two forms differ only by a short N-terminal sequence whose presence or absence is determined by differential translation initiation at two “in frame” ATG codons. (Gillman et al., Mol & Cell Biol 11: 2382-90 (1991)). The gro-1 open reading frame also contains two ATG codons at comparable positions, with the coding sequence between the two codons constituting a plausible mitochondrial sorting signal (FIGS. 3 and 5). It is likely therefore that DMAPP transferase in worms also exists in two forms, mitochondrial and cytoplasmic.
It should be noted, however, that the sequence of hgro-1 shows only one in-frame methionine before the conserved ATP/GTP binding site (FIG. 9) As we cannot be assured to have determined the sequence of the full length transcript, it is possible that further 5′ sequence might reveal an additional methionine. Alternatively, in humans, the mechanism by which the enzyme is targeted to several compartments might not involved differential translation initiation. In this context, it should be noted that the sorting signals which can be predicted from the sequence of hgro-1p are predicted to be highly ambiguous by the prediction program PSORT II. Furthermore, a conceptual translation of the Drosophila sequence (AA816785) predicts only one initiator methionine before the ATP/GTP binding site as well as several in-frame stop codons upstream of this start (FIG. 10), suggesting that no additional upstream ATG could serve as translation initiation site. In the figure, stop codons are indicated by stop, methionines are indicated by Met, and the conserved ATP/GTP binding site is underlined.
Expression Pattern of GRO-1
We have also constructed a reporter gene expressing a fusion protein containing the entire GRO-1 amino acid sequence fused at the C-terminal end to green fluorescent protein (GFP); The promotor of the reporter gene is the sequence upstream of gop-1 (FIGS. 13A-13C), the first gene in the operon (see FIG. 4). The promotor sequence is 306 bp long starting 32 nucleotides upstream of the gop-1 ATG. It is fused at the exact level upstream of gro-1 where trans-splicing to SL2 normaly occurs.
The genes gop-2 (FIG. 14) and gop-3 (FIGS. 15A-15B) are also located in the operon (see FIG. 4), the second and third genes in the operon.
We first construct the clone pMQ8 in which gro-1 is directly under the promoter for the whole operon using the hybrid primers SHP160 (SEQ. ID. NO, 38) and SHP159 (SEQ. ID. NO:37) and the flanking primers SHP161 (SEQ. ID. NO:39) and SHP162 (SEQ. ID. NO:40) in sequential reactions each followed by purification of the products and finally cloning into pUC18 (FIG. 11).
Primers SHP151, (SEQ. ID. NO:36) and SHP170 (SEQ. ID. NO:44) where then used to amplify part of the insert in pMQ8 and clone in pPD95.77 (gift from Dr Andrew Fire) which was designed to allow a protein of interest to be transcriptionally fused to Green Fluorescent Protein (GFP) (FIG. 12).
The reporter construct fully rescues the phenotype of a gro-1(e24.00) mutant upon injection and extrachromosomal array formation, indicating that the fusion to the GFP moiety does not significantly inhibit the function of GRO-1. Fluorescent microscopy indicated that gro-1 is expressed in most or all somatic cells. Furthermore, the GRO-1::GFP fusion protein is localized in the mitochondria, in the cytoplasm as well as in the nucleus.
The Hap-1 Gene Product (FIG. 16)
hap-1 is homologous: to the yeast gene HAM1 as well as to sequences in many organisms including bacteria and mammals (FIG. 7).
The origin of the worm and yeast sequence is as described above and below. The human, sequence was inferred from a cDNA sequence assembled from expressed sequence tags (ESTs); the accession numbers of the sequences used were: AA024489, AA024794, AA025334, AA026396, AA026452, AA026502, AA026503, AA026611, AA026723, AA035035, AA035523, AA047591, AA047599, AA056452, AA115232, AA115352, AA129022, AA129023, AA159841, AA160353, AA204926, AA226949, AA227197 and D20115. The E. coli sequence is a predicted gene (accession 1723866).
Mutations in HAM1 increase the sensitivity of yeast to the mutagenic compound 6-N-hydroxylaminopurine (HAP), but do not increase spontaneous mutation frequency (Nostov et al., Yeast 12: 17-29 (1996)). HAP is an analog of adenine and in vitro experiments suggest that the mechanism of HAP mutagenesis is its conversion to a deoxynucleoside triphosphate which is incorporated ambiguously for dATP and dGTP during DNA replication (Abdul-Masih and Bessman, J Biol Chem 261 (5): 2020-2026 (1986)). The role of the Ham1p gene product in increasing sensitivity to HAP remains unclear.
Explaining the Pleiotropy of MiaA and Gro-1
Mutations in miaA, the bacterial homologue of gro-1, show multiple phenotypes and affect cellular growth in complex ways. For example, in Salmonella typhimurium, such mutations result in 1) a decreased efficacy of suppression by some suppressor tRNA, 2) a slowing of ribosomal translation, 3) slow growth under various nutritional conditions, 4) altered regulation of several amino acid biosynthetic operons, 5) sensitivity to chemical oxidants and 6) temperature sensitivity for aerobic growth (Ericson and Björk, J. Bacteriol. 166: 1013-1021 (1986); Blum, J. Bacteriol. 170: 5125-5133 (1988)). Thus, MiaAp appears to be important in the regulation of multiple parallel processes of cellular physiology. Although we have not yet explored the cellular physiology of gro-1 mutants along the lines which have been pursued in bacteria, the apparently central role of miaA is consistent with our findings that gro-1, and the other genes with a Clk phenotype, regulate many disparate physiological and metabolic processes in C. elegans (Wong et al., Genetics 139: 1247-1259(1995); Lakowski and Hekimi, Science 272: 1010-1013 (1996); Ewbank et al., Science 275: 980-983 (1997)).
In addition to the various phenotypes discussed above, miaA mutations increase the frequency of spontaneous mutations (Connolly and Winkler,

    
    
        J Bacteriol 173(5): 1711-21 (1991); Connolly and Winkler, J Bacteriol 171: 3233-46 (1989)). As described in the previous section we have preliminary evidence that gro-1(e2400) also increases the frequency of spontaneous mutations in worms.
    
    


How can the alteration in the function of MDAPP transferase result in so many distinct phenotypes? Bacterial geneticists working with miaA have generally suggested that this enzyme and the tRNA modification it catalyzes have a regulatory function which is mediated through attenuation (e.g. Ericson and Björk, J. Bacteriol. 166: 1013-1021 (1986)). Attenuation is a phenomenon by which the transcription of a gene is interrupted depending on the rate at which ribosomes can translate the nascent transcript. Ribosomal translation is slowed in miaA mutants, and thus, through an effect on attenuation, could affect the expression of many genes whose expression is regulated by attenuation.
gro-1(e2400) also produces pleiotropic effects and, in addition, displays a maternal-effect, suggesting that it is involved in a regulatory process (Wong et al., Genetics 139: 1247-1259 (1995). However, attenuation involves the co-transcriptional translation of nascent transcripts, which is not possible in eukaryotic cells were transcription and translation are spatially separated by the nuclear membrane. If the basis of the pleiotropy in miaA and gro-1 is the same, then a mechanism distinct from attenuation has to be involved. Below we argue that this mechanism could be the modification by DMAPP transferase of adenine residues in DNA in addition to modification of tRNAs.
A Role for Gro-1 in DNA Modification?
We observed that gro-71 can be rescued by a maternal effect, so that adult worms homozygous for the mutation, but issued from mother carrying one wild type copy of the gene display a wild type phenotype, in spite of the fact that such adults are up to 1000 fold larger than the egg produced by their mother. It is unlikely that enough wild type product can be deposited by the mother in the egg to rescue a adult which is 1000 times larger. This observation suggests therefore that gro-1 can induce an epigenetic state which is not altered by subsequent somatic growth. One of the best documented epigenetic mechanisms is imprinting in mammals (Lalande, Annu Rev Genet 30: 173-196 (1996)) which is believed to rely on the differential methylation of genes (Laird and Jaenisch, Annu Rev Genet 30: 441-464; Klein and Costa, Mutat Res 386: 103-105 (1997)). Modification of bases in DNA have also been linked to regulation of gene expression in the protozoan Trypanosoma brucei. The presence of beta-D-glucosyl-hydroxy-methyluracil in the long telomeric repeats of T. brucei correlates with the repression of surface antigen gene expression (Gommers-Ampt et al., Cell 75: 112-1136 (1993); van Leeuwen et al., Nucleic Acids Res 24: 2476-2482 (1996)).
gro-1 and miaA increase the rate of spontaneous mutations, which is generally suggestive of a role in DNA metabolism, and can be related to the observation that methylation is linked to spontaneous mutagenesis, genome instability, and cancer (Jones and Gonzalgo, Proc. Natl. Acad. Sci. USA, 94: 2103-2105 (1997)).
Does gro-1 have access to DNA? Studies with mod5, the yeast homologue of gro-1, have shown that there are two forms of Mod5p, one is localized to the nucleus as well as to the cytoplasm, and the other form is localized to the mitochondria as well as the cytoplasm (Boguta et al., Mol. Cell. Biol. 14: 2298-2306 (1994)). The nuclear localization is striking as isopentenylation of nuclear-encoded tRNA is believed to occur exclusively in the cytoplasm (reviewed in Boguta et al., Mol. Cell. Biol. 14: 2298-2306 (1994)). Furthermore, studies of a gene maf1 have shown that when mod5 is mislocalized to the nucleus, the efficiency of certain suppressor tRNA is decreased, an effect known to be linked to the absence of the tRNA modification (Murawski et al., Acta Biochim. Pol. 41: 441-448 (1994)). Finally, as described in the previous section, gro-1 contains a zinc finger, a nuclei acid binding, motif. The zinc finger could bind tRNAs, but as it is in the C-terminal domain of gro-1 and human hgro-1 that has no equivalent in miaA, it is clearly not necessary for the basic enzymatic function. We speculate that it might be necessary to increase the specificity of DNA binding in the large metazoan genome. It should also be noticed that the second form of Mod5p which is localized to mitochondria also has the opportunity to bind and possibly modify DNA as it has access to the mitochondrial genome. See the previous section entitled “A role for gro-1 in a central mechanism of physiological coordination” for an alternative possibility as to the function of GRO-1 in the nucleus.
MiaA and Gro-1 are Found in Complex Operons
We have found that gro-1 is part of a complex operon of five genes (FIG. 4). It is believed, that genes are regulated coordinately by single promoters when they participate in a common function (Spieth et al., Cell 73: 521-532 (1993)). In some cases, this is tit well documented. For example, the proteins LIN-15A and LIN-15B which are both required for vulva formation in C. elegans, are unrelated products from two genes transcribed in a common operon (Huang et al., Mol Biol Cell 5 (4): 395-411 (1994)) One of the genes in the gro-1 promoter is hap-1, whose yeast homologue has been shown to be involved in the control of mutagenesis (Nostov et al., Yeast 12: 17-29 (1996)). Under the hypothesis that gro-1 modifies DNA, it suggest an involvement of hap-1 in this or similar processes. The presence in the same operon also suggest that all five genes might collaborate in a common function. The phenotype of gro-1 suggests that this function is regulatory. In this context, it should be noted that miaA also is part of a particularly complex operon (Tsui and Winkler, Biochimie 76: 1168-1177 (1994)), although, except for miaA/gro-1, there are no other homologous genes in the two operons.
A Role for Gro-1 in a Central Mechanism of Physiological Coordination
We have speculated that the genes with a Clk phenotype might participate in a central mechanism of physiological coordination, probably including the regulation of energy metabolism clk-1 encodes a mitochondrial protein (unpublished observations), 4and its homologue in yeast has also been shown to be mitochondrial (Jonassen, T (1998) Journal of Biological Chemistry 273: 3351-3357). The yeast clk-1 homologue is involved in the regulation of the biosynthesis of ubiquinone (Marbois, B. N. and Clarke, C. F. (1996) Journal of Biological Chemistry 271: 2995-3004). Ubiquinone, also called coenzyme Q, is central to the production of ATP in mitochondria., In worms, however, we have found that clk-1 is not strictly required for respiration. How might gro-1 fit into this picture?
One link is that dimethylallyldiphosphate is known to be the precursor of the lipid side-chain of ubiquinone. In bacteria, ubiquinone is the major lipid made from DMAPP. In eukaryotes cholesterol and its derivatives are also made from DMAPP. Interestingly, C. elegans requires cholesterol in the growth medium for optimal growth. This link, however, remains tenuous in particular in the absence of an understanding of the biochemical function of CLK-1.
In several bacteria, the adenosine modification carried out by DMAPP transferase is only the first step in a series of further modification of this base (Persson et al., Biochimie 76: 1152-1160 (1994)). These additional modifications have been proposed to play the role of a sensor for the metabolic state of the cell (Buck and Ames, Cell 36: 523-531 (1984); Persson and Björk, J. Bacteriol. 175: 7776-7785 (1993)). For example, one of the subsequent steps, the synthesis of 2-methylthio-cis-ribozeatin is carried out by a hydroxylase encoded by the gene miaE. When the cells lack miaE they become incapable of using intermediates of the citric acid cycle such as fumarate and malate as the sole carbon source.
Another link to energy metabolism springs from the recent biochemical observations of Winkler and coworkers using purified DMAPP transferase (E. coli MiaAp) (Leung et al., J Biol Chem 272: 13073-13083 (1997)). These investigators observed that the enzyme in competitively inhibited by phosphate nucleotides such as ATP or GTP. Furthermore, using their estimation of Km of the enzyme and its concentration in the cell, they calculate that the level of inhibition of the enzyme in vivo, would exactly allow the enzyme to modify all tRNAs but any further inhibition would leave unmodified tRNAs. This suggests that the exact level of modification of tRNA (or of DNA) could be exquisitely sensitive to the level of phosphate nucleotides. Superficially, this is consistent with the phenotypic observations. The state of mutant cells which lack DMAPP transferase entirely would be equivalent of cells where very high levels of ATP would completely inhibit the enzyme. Such cells might therefore turn down the ATP generating processes in response to the signal provided by undermodified tRNAs (or DNA).
More generally, GRO-1 could act in the crosstalk between nuclear and mitochondrial genomes. The nuclear and mitochondrial genomes both contribute gene products to the mitochondrion energy-producing machinery and these physically separate genomes must therefore exchange information somehow to coordinate their contributions (reviewed in Poyton, R. O and McEwen J. E. (1996) Annu. Rev. Biochem. 65: 563-601) Furthermore, the energy producing activity of the mitochondria is essential to the rest of the cell, and the needs of a particular cell at a particular time must be somehow convey to the organelle to regulate its activity. GRO-1 could participate in this coordination in the following manner. GRO-1 is found in three compartments, the nucleus, the cytoplasm and the mitochondria (see above), and thus has the opportunity to regulate gene expression in more that one way. How could its action coordinate gene expression between compartment? GRO-1 could partition between the mitochondria and the nucleus and its relative distribution could be determined by the amount of RNA (or mtDNA) in the mitonchodria (Parikh, V. S. et al. (1987) Science 235: 576-580). For example, if the cell is rich in mitochondria, much GRO-1 will be bound there which could result in a relative depletion of activity in the cytoplasm with regulatory consequences on the translation machinery. Binding of GRO-1 in the nucleus could have similar consequences and provide information about nuclear gene expression to the translation machinery.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

## Claims

1. An isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO:3, or the complete complement thereof.

2. An isolated polynucleotide that encodes a polypeptide comprising:
(i) the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3, or 
(ii) the amino acid sequence of SEQ ID NO: 63; or the complete complement of said polynucleotide.

3. The polynucleotide of claim 1 or 2 further comprising one or more nucleic acid sequences encoding a heterologous polypeptide.

4. The polynucleotide of claim 3 wherein the heterologous polypeptide is green fluorescent protein (GFP).

5. A vector comprising the polynucleotide of claim 1 or 2.

6. The vector of claim 5 that is an expression vector.

7. An isolated host cell which comprises the vector of claim 5.

8. An isolated host cell which comprises the vector of claim 6.