Stein JA et al. (1984) Worm Breeder's Gazette "a computer program for organizing a clone collection."

Emmons SW, Stein JA

[Worm Breeder's Gazette, 1984]

We have written a computer program for storage and retrieval of information about the recombinant DNA collection held by a laboratory. For each clone, information such as clone name, cloning vector, cloner, date cloned, notebook page, storage location, etc., is entered. The cloned insert is identified by a serially-assigned fragment number and by a restriction map. Wherever a given fragment occurs in the collection it is identified by its fragment number, and all restriction mapping data for that fragment is held together in a single storage location and kept updated. Data regarding interrelationships among fragments in the collection, such as which are subclones of others, are also held. The stored information can be searched and listed in a variety of ways. The program is modular in design and can be readily modified and expanded. It is written in Basic for a Dec RT-11 operating system ( PDP-11 computer or equivalent) and should be easily adaptable to other systems. We encourage other laboratories to use it. The program anticipates multiple laboratory use by prefixing each fragment number with a laboratory number. By this means each cloned segment of the genome is given a unique name. Eventually there will undoubtedly be a need for a centralized listing of information about cloned fragments of the C. elegans genome. We suggest that a centralized listing could be a subset of the information stored in laboratory based listings such as this one. For a free interchange of information among laboratories and between laboratories and a centralized data base to be possible, a uniform system of nomenclature and computerized format will have to be adopted. We have written this program in order to gain some experience with the use of a laboratory based data bank that hopefully will be helpful in designing a widely used system. Please contact us if you are interested in using this program. We would be glad to help you adapt it to your computer system.

Forrester WC et al. (1995) Worm Breeder's Gazette "Making Worms Sit Still on Plates."

Forrester WC, Garriga G

[Worm Breeder's Gazette, 1995]

Making worms sit still on plates Wayne Forrester and Gian Garriga. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. We sought to anesthetize C. elegans on culture plates to allow examination of green fluorescent protein expression by fluorescence microscopy at high magnification using a long focal length objective. We tested carbon dioxide as an anesthetic because it is effective in several organisms. Wild-type worms held under a gentle stream of CO2 stopped moving. The response was very rapid; when a stream of CO2 was placed over the animals they stopped almost immediately. Similarly, animals recovered within a few seconds of removing the CO2. The very high tech apparatus we used consisted of a stoppered 1 liter side-arm flask containing ~15 pieces (2-3 cm diameter) of dry ice, and flexible hose attached to the side-arm. CO2 sublimation forced a gentle stream of gas through the tubing, which was held a few centimeters above the surface of the plate. We presume that the effect is due to CO2. Alternatively, nematodes might doze when subjected to a gentle breeze or in response to a slight drop in temperature. We performed several carefully controlled experiments to determine whether either of these alternate explanations was plausible. To determine whether worms arrest in response to a breeze, we applied a gentle stream of air using the house air supply. When this was held over a plate of N2, many animals initially crawled backwards, perhaps due to stimulation of mechanosensory neurons, but then resumed normal locomotion. Therefore, a gentle stream of air does not induce nematode napping. To determine whether the effect was due to lower temperature, we performed two experiments. First, we determined that heating the CO2 stream to 26C, which was 3C warmer than the standard set-up, did not abolish the effect; worms still stopped moving. Second, we determined that chilling air to 21.5C did not anesthetize worms. Thus a stream of cool air does not cause C. elegans slumber, but a stream of warm CO2 does. In conclusion, we find that nematodes on plates can be anesthetized by placing them continuously in a gentle stream of CO2. Anesthetizing nematodes on plates could facilitate counting animals, scoring phenotypes that are difficult to see in active worms, and viewing worms on plates by fluorescence microscopy. We hope others will find this observation useful.

Albertson DG (1988) Worm Breeder's Gazette "some observations on meiosis."

Albertson DG

[Worm Breeder's Gazette, 1988]

The meiotic chromosomes of C. elegans, in common with those of other animals and plants with holocentric chromosomes, appear to lack any recognizable kinetochore or centromere region. In organisms with monocentric chromosomes the centromere serves two functions in meiosis; to hold the half bivalents together until anaphase II and to attach the chromosome to the meiotic spindle for disjunction. In certain insects where extensive cytological studies of meiosis have been made, the chromatids of the meiotic bivalent are held together in an end-to- end association. They are oriented parallel to the equatorial plate ( equatorial orientation) and microtubules appear to project into the chromatin. Chiasmata which have terminalized by diplotene-diakinesis hold the half bivalent together until anaphase II. Some years ago Nichol Thomson and I investigated the behavior of C. elegans chromosomes in meiosis by reconstructing meiotic spindles from serially sectioned male and hermaphrodite gonads. We observed that in C. elegans each half bivalent lies just above or below the equatorial plate and that the half bivalents are held together by a chromatin thread. Microtubules appeared to project into the chromatin. The small size of the chromosomes made it impossible to determine whether the homologues were associated end-to-end in diakinesis or whether the chromosomes were aligned in an equatorial or axial (long axis of the chromosome perpendicular to the plate) orientation on the plate. However, it appears from cytological observations that the chromosomes probably are associated end-to-end in diakinesis. Translocation of part of the X chromosome to the right arm of LGI in mnDp10 and mnDp25 chromosomes resulted in cytologically visible knobs at either end of the bivalent in diakinesis. Furthermore in heterozygotes the knobs were seen on only one end of the bivalent (Herman et al., 1979). Similarly, in mnT2(X;II) hermaphrodites the bivalent consisting of LGII and mnT11(X;II) is asymmetric, one half bivalent being longer than the other at diakinesis. Recently, I have looked at the orientation of the meiotic bivalents again, but this time have used in situ hybridization to label the ends of LGI and LGII. A biotin labeled ribosomal DNA probe was hybridized with 'whole mounts' of N2 or let(e2000);eDp20(I;II) gonads and embryos and the site of hybridization, detected by immunofluorescence was visualized by two-color confocal microscopy so that the fluorescently stained chromatin could be precisely aligned with the hybridization signal. At diakinesis the ribosomal probe was observed on the outside ends of both LGI and LGII (eDp20) bivalents. It has also been possible to make a few observations on the orientation of the bivalents at meiosis I and in these cases the ribosomal probe was seen on the ends of the chromosome pointing towards the spindle poles. Therefore in C. elegans the bivalents orient axially at meiosis I and for LGI and LGII the right end of the chromosome leads to the spindle pole, while the bivalent is held together until anaphase I by the end- to-end association of the left ends of these homologues. It is possible that the chromatin threads seen extending across the equatorial plate in the electron microscope are terminalized chiasmata that serve to hold the half bivalents together at meiosis I. These observations suggest that the two functions of the meiotic centromere are divided between the two ends of the chromosome in C. elegans. One end takes on the function of holding the bivalent together until anaphase I (rather than anaphase II) possibly by terminalized chiasmata, while the other end may be involved in spindle attachment. Since the bivalents are oriented axially, the first meiotic division is reductional, causing associations through terminalized chiasmata to be broken at anaphase I. Therefore another mechanism is required to hold the half bivalents together at metaphase II.

Mackenzie JM et al. (1977) Worm Breeder's Gazette "structural studies of nematode body-wall muscles."

Epstein HF, Mackenzie JM

[Worm Breeder's Gazette, 1977]

We have pursued detailed studies of the structure of N2 body-wall muscle as a necessary condition for deciphering the abnormalities of various sarcomere-defective mutants, particularly many which have more subtle defects than unc-54 mutants, and also to understand structural changes in body-wall muscle cells during development. Using polarized light microscopy, we find that the body wall muscle cells in relaxed, anesthetized N2 animals are arranged right to left +/-11.9 to the anterior-posterior axis. The sarcomeres within these cells are arranged +/- 5.9 to the same axis. Under these conditions, the sarcomere width normal to the cell is 1.67 and the similarly measured A band width is 1.0 . By both polarized light and electron microscopy we find that the orientation of the thick filaments is parallel to the N2 anterior-posterior axis. Thus, from these observations, the length of the thick filaments is calculated to be 9. 7 , assuming that all the thick filaments are held in register. It would be difficult to accurately measure the length of such very long filaments by electron microscopy of thin sectioned material. In roller mutants such as the dominant SU1006 where the cell orientation is significantly different from N2 and variable, all the internal relationships and dimensions are preserved. Similarly in dumpy mutants such as E61, although cell lengths are shortened, all other features such as orientation and internal dimensions are similar to N2.

Avery L (1989) Worm Breeder's Gazette "functions of the GREENs: slippery pharynxes and serotonin."

Avery L

[Worm Breeder's Gazette, 1989]

In my initial survey of the pharyngeal neurons, I classified them into three groups: M4, MC, and the 12 remaining anatomical types of pharyngeal neurons, which I call the GREENs. None of the individual GREEN neuron types is necessary for superficially normal pumping, but when all 12 are killed, pumping becomes very abnormal. I have recently made some progress in figuring out what they are doing. During normal pumping, bacteria move posterior in the pharynx when the muscles contract, and are held where they are when the muscles relax again, so that they are efficiently transported towards the intestine. When all the GREENs were killed, the pharynx appeared to become slippery: bacteria slid anterior as well as posterior. When bacteria were abundant, the lumen of a GREEN pharynx became distended with them, and they were ground up and passed to the intestine only slowly. Seymour et al (J Zool 201: 527) showed by microcinematography that in normal pumping the contraction and relaxation of the corpus muscles are not precisely simultaneous along its length: both begin at the front. They proposed this has a function in trapping bacteria in the pharynx. I suspect that in the absence of the GREENs this fine timing goes awry, and as a result bacteria are not held back by the relaxing pharyngeal muscles. The GREEN neuron I understand best at present is M3. When M3 was killed, there was a subtle abnormality in pumping: the contractions lasted longer. Conversely, when all the GREENs except M3 were killed, contractions were very brief. Thus M3 hastens pharyngeal muscle relaxation during a pump, and is opposed by some other(s) of the GREENs. I5 is probably (one of) the GREEN(s) that opposes M3, since pumps became briefer when I5 alone was killed. When both M3 and I5 were killed, pumps were long, just as when M3 alone was killed, suggesting I5 may exert its effects on pump length via M3. In fact, I5 does synapse on M3. There was also a new effect when both M3 and I5 were killed: the isthmus became slippery. The timing of anterior isthmus contraction was abnormal in M3-I5 worms: it contracted just after the corpus contraction, instead of simultaneously with it, and remained contracted longer. M3 is also important for corpus function. In fact, when all the GREENs except M3 were killed, the corpus was not slippery. Other GREENs important for normal corpus function are I2, I6, M1, and possibly I4. M5 may also be important for isthmus function. However, I have not worked out the details for any of these. I have also learned something about GREEN function in a completely different way. I discovered previously that imipramine stimulates pumping in wild-type worms, but not in mutants that have reduced levels of serotonin (WBG 10(2): 39). The only serotonergic neuron in the pharynx is NSM, and the only pharyngeal neurons connected to the extrapharyngeal nervous system are I1 and M1. I therefore killed NSM, I1, and M1, thinking to remove any serotonin from the pharynx, and to sever its communication with extrapharyngeal serotonergic neurons that might be stimulating pumping. Sure enough, imipramine did not stimulate pumping in I1- M1- NSM- worms. I have not yet tested whether it is necessary to kill all three to get the effect.

Hodgkin JA (1988) Worm Breeder's Gazette "wild isolates and mating plug formation."

Hodgkin JA

[Worm Breeder's Gazette, 1988]

Many natural isolates of C. elegans differ from the standard laboratory strain, N2, in copulatory plug formation: the males from these strains deposit a gelatinous blob over the vulva of a mated hermaphrodite, but N2 males do not. Previously (Hodgkin, Doniach & Kenyon, WBG 8-3:36) we showed that the plugging trait, as discovered in the wild isolate Sta-5 (formerly named Cal-5) behaved as a Mendelian dominant, mapping to the locus plg-1 on LGIII. We suggested that N2 had lost the plugging trait at some point during its scientific career. A survey of natural isolates indicates that this is not the case; instead, it appears that many natural races are nonpluggers. Male stocks were established from the 24 strains listed in the table, and males were tested for plug formation. All non- plugging strains were crossed with N2, and F1 heterozygous males tested: all were negative. Thus, if non-plugging represents loss-of- function, then all these strains carry recessive mutations of the same gene, plg-1. Also, the plugging trait in two of the positive strains, AB3 and RC301, was examined genetically, by crossing with N2-derived mapping strains. In both cases the trait behaved as a dominant mapping to approximately the same location as plg-1.In almost all of these races (or at least the stocks of them presently held in Cambridge) males are present at less than 1% of the individuals in a growing population, but these males are always potent and male stocks are easily established. The only exceptions are the two Bergerac strains, RW7000 and N62, which are well known to produce males that mate very poorly. It is not obvious what advantage or disadvantage is associated with plugging. The trait is stable in culture: both plugging and non- plugging strains maintain their character over hundreds of generations.

Hartman PS et al. (1983) Worm Breeder's Gazette "developmental regulation of DNA repair in C elegans."

Lowery K, Hartman PS

[Worm Breeder's Gazette, 1983]

Synchronized N2 populations were obtained by growth on chicken egg plates, followed by hypochlorite treatment. Embryos were plated on seeded NG plates, removed at various times, and subjected to UV irradiation. After incubation in phosphate buffered saline to allow time for repair, worms were frozen in liquid nitrogen. The DNA was extracted and treated with a UV-specific endonuclease, partially purified from Micrococcus luteus, which introduces single strand breaks between adjacent nucleotides of pyrimidine dimers. The number of single-strand breaks, and hence the number of dimers present in the DNA, was measured in two ways. First, samples were subjected to alkaline gel electrophoresis. Weighted average molecular weights were calculated by comparison with molecular weight standards. Second, samples were incubated with DNA polymerase I and supplied with all factors necessary for polymerization, including tritiated deoxycytidine. The amount of radioactive incorporation was proportional to the number of dimers. The techniques yielded similar results. Repair was evident in animals irradiated 24 hours after hypochlorite treatment (as L1's), with a maximum of 70% to 90% of the dimers removed 24 hours after irradiation. The number of dimers did not change when either L4 larvae or adults were held after irradiation. In fact, in most experiments, DNA from adults was partially degraded after irradiation. The simplest conclusion is that DNA repair is developmentally regulated in the worm; specifically, young larvae have substantial ability to excise DNA damage but this capacity diminishes throughout development. This regulation correlates well with the animal's cellular development,in which many somatic cells undergo the terminal round of DNA replication during larval development. Taken together, our results suggest that dpy-21+ is a negative regulator and dpy-22+ is a positive regulator of X-expression in both XO and XX animals. The finding that dpy-22(e652) is apparently epistatic to dpy-21(e428) in the double mutant suggests further that dpy-21+ may act by negatively regulating dpy-22. We have preliminary evidence that the one known dpy-23 mutation does not affect the three X-linked hypomorphs. We would like to have a larger collection of both X-linked and autosomal hypomorphs, and will welcome any contributions.

Hartman PS (1984) Worm Breeder's Gazette "effects of age and liquid holding on the UV radiation sensitivities of N2 and radiation-sensitive (rad) dauer larvae."

Hartman PS

[Worm Breeder's Gazette, 1984]

Wild-type (N2) and four radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, were not hypersensitive. Irradiation also resulted in a significant delay in development. For example, over 95% of the unirradiated N2 dauer larvae completed development within three days of food presentation; however, fluences of 78 Jm-2 and 156 Jm-2 resulted in average delays of approximately 40 and 50 hours, respectively. In fact, higher fluences resulted in developmental periods as long as 7.5 days. Brood sizes decreased in a fluence-dependent fashion; however, most N2 survivors which were delayed in development for up to four days still produced greater than 10 offspring per animal. The fact that dauer larvae do not resume development until placed in the presence of food was exploited to test two aspects of their radiation response. First, the effect of dauer age on radiation sensitivity was examined by irradiating N2 dauer larvae at 0, 25 or 50 days after harvesting. Animals were collected and either irradiated immediately or incubated in PBS for 25 or 50 days before irradiation. There were no significant differences in radiation sensitivities. Second, the effect of post irradiation holding on survival was tested on N2 dauer larvae. Animals were irradiated with either 116 or 194 Jm- 2 and incubated in a non-nutritive medium (PBS) for various intervals before exposure to food. Liquid holding recovery (LHR) refers to the increased survival observed in many instances when irradiated organisms are held in a nonnutritive medium before plating. Genetic and biochemical evidence in both bacteria and yeast indicate that increased survival can be attributed to DNA repair that occurs during holding. No liquid holding recovery (LHR) occurred at either fluence; in fact, survival decreased after approximately five days of post- irradiation holding. In addition, no differences were observed in the amount of developmental delay imposed by radiation. These results imply that this developmental state is not a period of active DNA repair.

Laufer JS et al. (1976) Worm Breeder's Gazette "the cuticle of C elegans."

Laufer JS, Edgar RS, Kusch M, Pillsbury A

[Worm Breeder's Gazette, 1976]

We are starting to study the structure and genetic control of the C. elegans cuticle. Cuticles free of most contaminating tissue can be obtained by gentle homogenization or sonication followed by extensive washing with or without SDS. Such cuticles reveal many structural features in phase contrast observation. Cuticle material not only covers the outer surface but continues into the anal opening and the lining of the pharynx. The pharynx apparently also has a lining of SDS-resistant material separating it from the body proper. The cuticle appears to be composed of two layers which are held together by 'struts'. The struts form a quite regular pattern consisting of a double row at the boundary between the annulae. Mild treatments with Pronase or Collagenase appear to act most readily by breaking the struts resulting in the formation of 'double-bags'. Continued treatment results in the dissolution of the inner 'bags'. In addition to being relatively resistant to enzymatic digestion, the outer layer is much more resistant to a variety of non-enzymatic treatments that break non-covalent or disulphide bonds. Adults and dauer larvae have the familiar cuticular structure with annulae and lateral treads ( ridges, alae), although the structure of the tread differs in the two forms. Other larval forms (L3, L4), not yet precisely characterized, lack these structural features. We are about to attempt chemical characterization of the isolated cuticle. The cuticles of dumpy, roller and blister mutants display abnormal cuticular features. In blister mutants the blisters form between the two cuticle layers with the struts clearly broken or missing. The cuticles removed from roller animals are helically twisted, in some cases these cuticles physically curl up even more after the animal is removed. The annulae are not arranged at right angles to the treads as in a normal animal. Mutants with an extreme roller phenotype, and some dumpies, have aberrant treads displaying multiple loops and breaks. These observations encourage us in our suspicion that at least some of the genes that generate these phenotypes are concerned with cuticle formation. Unsolicited gifts of newly isolated roller or blister mutants will be gratefully accepted.

Swanson MM (1982) Worm Breeder's Gazette "Caenorhabditis genetics center news."

Swanson MM

[Worm Breeder's Gazette, 1982]

1. We always welcome map data. Please send any crucial corrections to the current map immediately for inclusion in Volume II of Genetic Maps (O'Brien). 2. A list of 825 CGC strains with complete genotypic descriptions has been mailed recently to all labs doing C. elegans genetics. A list of the wild-type stocks in our collection is included. A limited number of lists are still available in case we missed a lab which needs this kind of information. 3. The CGC Computerized Data Retrieval System is fully functional. The system permits telephone-linked access to the data files. Recently we received NIH approval to implement the system, so we have distributed the user's manual to those of you who indicated an interest at Cold Spring Harbor. Users will be charged $10/hr for computer time (an unfortunate necessity at present). 4. Each lab using CGC services could help us in two ways: (1) State the proposed use of stocks requested from us in your letters. A single sentence noting the area of research and/or funding source is usually sufficient. (e.g., 'I plan to test these strains for enzyme activities for an NIA-funded project on factors correlated with lifespan.') (2) We would appreciate your care in remembering to acknowledge CGC services in all publications mentioning stocks furnished by the CGC. We must furnish copies of such articles to the NIA periodically. 5. Copies of the April, 1981, issue of the Genetic Map (distributed at the Cold Spring Harbor Meeting) are still available on request. 6. Included here are two bibliographic updates: (1) covering the period from the last newsletter to the meeting at Cold Spring Harbor, and (2) covering the period since the meeting. 7. At the request of Bob Edgar, a list of chromosomal rearrangements on the genetic map data also is included here. Each rearrangement is listed by linkage group and by name (alphabetical order). Those presently held in CGC strains are noted by an asterisk. Even more rearrangements may be found in the strain list mentioned above. 8. Finally, current lists of genetic designations (lab list and gene list) are included, to round out this fabulous package deal.