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[
Worm Breeder's Gazette,
1996]
We are studying genes that function in the nematode nervous system using a dauer formation phenotype. The nematode senses environmental cues, such as dauer pheromone, the presence/absence of food, and temperature, and makes a decision to form a dauer. Dauer formation phenotype reflects, at least in part, neuronal activities and can be used as an indicator of a nervous system function. We are particularly focusing on the
unc-31;
aex-3 dauer constitutive (Daf-c) phenotype (1). Single mutants of either
unc-31 or
aex-3 are not Daf-c. However,
unc-31 and
aex-3 double mutants are strongly Daf-c at 25C. We made various combinations of
unc-31 (
e169,
n928, and
u280) and
aex-3 (
ad418,
sa5, and
ad696) alleles. The weakest is
unc-31(
e169);
aex-3(
ad418), which is 65% Daf-c at 25C and 30% at 15C. The strongest is
unc-31(
u280);
aex-3(
ad696), which is 100% Daf-c at 25C and 95% at 15C.
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[
Worm Breeder's Gazette,
1990]
The C. elegans DNA sequence database is growing large enough to permit preliminary compilations and predictions which should be useful for analyzing new sequences. We extracted the DNA sequence flanking the ATG initiation codons from 54 C. elegans genes (34 were available in GenBank (Release 61.0)); after alignment they formed the matrix shown below, which yielded the consensus (A/c)A(a/c)(A/C)ATG (lower case implies weakly conserved). While this is obviously a small sample size the result is significantly different from the vertebrate consensus derived by Kozak(1) (N=699). The total information content in these two matrices(2, 3) differs by 1 bit (C. elegans ~7.6 bits ( genome=36% G+C), vertebrates ~8.6 bits (genome=40% G+C)), although the distributions are roughly homologous (see graph below; baseline ~0.06 bits). Because random sequences will contain many spurious homologies this consensus will be most useful for evaluating suspected initiation codons. The following sequences were used to generate the C. elegans matrix:
act-1,
act-3,
ama-1,
cal-1,
col-2,
col-7,
col-14,
deb-1, pd-2,
gpd-3,
gpd-4,
glp-1,
gyt-1,
his-1,
his-3,
his-9,
his-10,
hts-11,
hts-12,
hsp-1,
hsp-6,
hsp16-1,
hsp16-2,
hsp16-41,
hsp16-48,
mec-3,
lin-12,
msp-74,
myo-1,
myo-3,
unc-54,
vit-2,
vit-5, al msp sequences were not incorporated. If you are interested in increasing the utility of this 'ribosome binding site' (RBS) consensus please send us your sequences and we will incorporate them into the matrix and redistribute the results. [See Figure 1]
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[
Worm Breeder's Gazette,
1999]
In the past two decades much effort in biology has gone into cloning and sequencing of genes. The accomplishment of genome sequencing projects, such as that of Caenorhabditis elegans, is leading to a new scenario, where all genomic information is available, and the remaining challenge is to interpret it in biological terms. It is becoming essential to develop strategies to link non coding sequence to function. We have set up a one hybrid system kit, using C. elegans as model, to search the DNA for sites of binding of structural and regulatory proteins. We have constructed yeast strains in which C. elegans non-coding DNA sequences (baits) are inserted, in place of the UAS, upstream the two test genes HIS-3 and LacZ. The recipient strain is a diploid ura- yeast strain carrying a deletion of the HIS-3 gene on one chromosome and a wild type copy on the other. We have chosen to use diploid strains because the most frequent cause of HIS spontaneous reversion is the transposition of a Ty element upstream the HIS coding region; in diploid strains the transposable element Ty is quiescent. Our bait sequences are cloned upstream the HIS-3 gene in an integrative plasmid and transformed in yeast after restriction digestion. The integrative plasmid had been constructed by inserting a polylinker into the partially deleted HIS-3 promoter for ease of manipulation and it contains homology cassettes appropriate for gene replacement and to facilitate the screening of the integrants. It contains, in fact, HIS-3 flanking regions for proper integration of the plasmid, and the selectable marker URA3 in such a position to be deleted, after integration, via homologous recombination together with the wild type copy of HIS-3. Such event can be selected by plating the recombinant strains on FOA (5-fluor-orotic acid), only the ura- cells will growth in such conditions. Most pop-outs occur by recombination in the extensive tracts of homology between the recombinant HIS-3 sequence and the wild type. The obtained yeast strains are auxotrophic for histidin in the presence of only 5mM aminotriazol unless a protein binds to the bait sequence and activates transcription by mean of an activation domain such as that of GAL-4. This is the selection tool for the screening of our cDNA library (described in Ederle et al. European C. elegans Meeting 1996). In all cases the second test gene (LacZ) is carried as an episome by the cell. We have tested our tools by cloning the GATA-box as DNA bait and successfully screening our library. We are now proceeding in using this kit for the analysis of gene promoter regions and other DNA sequences potentially target of binding proteins. 1) Ederle S, De Felice B, Pulitzer JF, La Volpe A (1996) A C. elegans cDNA library for one hybrid/two hybrid system screening in yeast. 2nd European C. elegans Meeting Mont Saint-Odile, France. p.23.
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[
Worm Breeder's Gazette,
1991]
Ed Hedgecock came to Tokyo in January for the 2nd seminar on Molecular and Developmental Neurobiology and his cell migration work impressed scientists of other animal field. Sydney Brenner won the Kyoto Prize and came to Japan on 23 of October for receiving the Prize on his fourth visit in this year. He talked with his excellent joke about telescope in Astronomy and microscope in Biology. Sydney is the man who gave the main lecture in the First Meeting of Japan Molecular Biology Society. Iva Greenwald came to Japan for attending the Naito Foundation International Workshop on Morphogenesis Program in early November together with her fiance Gary Struhl. It is true that the relation between Drosophila and Caenorhabditis is very good in U.S. and Japan. Some nematode scientists in Japan got grant from Drosophila project. John Sulston gave the lecture entitled 'The Genome of Caenorhabditis' on 28th of November at the main invited lecture of the Thirteenth Meeting of Japan Molecular Biology Society in Kyoto International Congress Hall. The lecture impressed many Japanese scientists especially his 'The Logical Next Step: Genome Sequencing'. On the 27th night, 34 worm people assembled to the worm party for welcoming to him. Sixteen papers from five labs were presented at the meeting.
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[
Worm Breeder's Gazette,
1978]
The brownish-yellow bacterium that commonly contaminates worm stocks is Pseudomonas aeruginosa. It is sensitive to gentamicin and colistin, we use gentamicin (10 g/ml) in some plates to control it. When present on plates, it reduces progeny production substantially and also causes rapid destruction of unfertilized oocytes. The bulk E. coli commercially available from Grain Processing Corp. can be grossly contaminated with Pseudomonas and other species. (When asked if they could prepare E. coli aseptically, the reply was 'No, the man that harvests the pellets scoops them up with his bare hands'. On special request he will wash his hands). After receiving a large batch of bacteria which would not grow worms well, we switched to growing our own E. coli in a Lab-Line High density fermentor.
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[
Worm Breeder's Gazette,
2002]
The purpose of this study was to investigate the effect of different concentrations of ascorbic acid in water solutions on nematode life span. In this experiment ascorbic acid was used in following dilutions: 1:10 1 , 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 and 1:10 7 . Three adult animals (3 5 days old) were kept in microtitre wells containing 0,5 ml of liquid medium (with E. coli and without ascorbic acid) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with ascorbic acid in any concentration) every day (one worm in one well) beginning from third day. This investigation was carried out in temperature +21 0 C and in the darkness. The obtained results are presented in the following table. Concentration of ascorbic acid n Longevity (days) Mean +/- S.E. Maximal Control 12 13,7 +/- 1,6 28 1:10 1 12 toxic 1:10 2 12 15,4 +/- 1,3 23 1:10 3 12 17,9 +/- 1,6 22 1:10 4 12 22,0 +/- 0,7 29 1:10 5 12 18,6 +/- 1,3 30 1:10 6 12 19,3 +/- 1,3 30 1:10 7 12 16,3 +/- 1,1 27 Conclusion: If ascorbic acid solution was applied to C. elegans , it was able to increase their mean (by 61,0%, p<0,001) as well as maximal longevity in comparison with control in dilution of 1:10 4 . Acknowledgment: The author wish es to express his thanks to CGC for providing C. elegans (Bristol, N2) and E. coli OP50.
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[
Worm Breeder's Gazette,
1996]
The purpose of this study was the effect of different concentrations of royal jelly extract on nematode life span. In this experiment royal jelly extract was used in the form of "Apilactosa" (produced by "Fertane", Moscow, Nagatinskaya str., 1) in following dilutions 1:10*3, 1:10*4, 1:10*5, 1:10*6 , 1:10*7 , 1:10*8 , 1:10*9 and 1:10*10. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0,75 ml of liquid medium (with E. coli and without royal jelly extract) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with royal jelly extract in any concentration) every day (one worm in one well). This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Concentration of royal Longevity jelly extract n mean +/- S.D. maximal 1:10*3 12 Lethal 1:10*4 11 18,2 +/- 1,8 28 1:10*5 12 19,2 +/- 2,2 32 1:10*6 12 19,3 +/- 1,7 28 1:10*7 12 15,8 +/- 2,2 28 1:10*8 11 20,6 +/- 3,1 32 1:10*9 8 12,3 +/- 3,5 34 1:10*10 12 17,5 +/- 2,3 31 Conclusion: If the extract from royal jelly was applied to C. elegans during the whole life span in above described conditions, the most effective concentration was 1:10*8. It seemed for me more reasonable to study the effect on longevity of any concentrations near 1:10*8. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (Bristol, N2) and E. coli OP50.
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[
Worm Breeder's Gazette,
1999]
the effect of different concentrations of procaine hydrochloride in water solutions on nematode life span. In this experiment procaine hydrochloride was used in following dilutions: 1:10^3, 1:10^4, 1:10^5 and 1:10^6. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0.75 ml of liquid medium (with E. coli and without procaine hydrochloride) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with procaine hydrochloride in any concentration) every day (one worm in one well) beginning from third day. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Concentration of procaine hydrochloride n Longevity (days) Mean + S.E. Maximal Control 24 9.7 + 0.6 15 1:10^3 24 11.3 + 0.8 20 1:10^4 24 11.3 + 1.0 25 1:10^5 24 14.3 + 1.9 38 1:10^6 24 11.2 + 0.9 21 Conclusion: If procaine hydrochloride solution was applied to C. elegans, it was able to increase their mean longevity [by 47.4 per cent, statistically significant (P < 0.05)] in dilution 1:105. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (Bristol, N2) and E. coli OP50.
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[
Worm Breeder's Gazette,
2001]
The purpose of this study was to investigate the effect of different concentrations of sodium fusidine in water solutions on nematode life span. In this experiment sodium fusidine was used in following dilutions: 1:10 3 ,1:10 4 ,1:10 5 ,1:10 6 ,1:10 7 and 1:10 8 . Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0,75 ml of liquid medium (with E. coli and without sodium fusidine) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with sodium fusidine in any concentration) every day (one worm in one well) beginning from third day. This investigation was carried out in temperature +21 0 C and in the darkness. The obtained results are presented in the following table. Concentration of sodium fusidine n Longevity (days) Mean +/- S.E. Maximal Control 36 9,47 +/- 0,76 21 1:10 3 36 10,03 +/- 0,81 23 1:10 4 36 9,44 +/- 0,88 25 1:10 5 36 8,72 +/- 0,51 18 1:10 6 36 9,86 +/- 0,94 26 1:10 7 36 8,86 +/- 0,73 24 1:10 8 36 8,86 +/- 0,73 15 Conclusion: If sodium fusidine solution was applied to C. elegans, it was not able to increase their mean as well as maximal longevity in comparison with control. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (Bristol, N2) and E. coli OP50.
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[
Worm Breeder's Gazette,
1996]
Exactly 100 years ago (Jan 10, 1896) Edwin Conklin published a paper entitled "Cell Size and Body Size." In it he posed the following question: do species that vary in adult body size differ in cell number or in cell size? His answer, for a genus of intertidal snails, was that species that vary in body size vary in cell number, but that cell size is more or less constant over evolutionary time.