Johnson TE (1980) Worm Breeder's Gazette "Generation of X-linked Mosaics"

Johnson TE

[Worm Breeder's Gazette, 1980]

We are investigating somatic loss of X-chromosomes as a means of producing mosaic worms at relatively high frequencies. We produce worms heterozygous for the X-linked muscle morphology mutant, unc-78( e1217), which are homozygous for various him's. Only one such mutant, him-2(e1065), generates spontaneous mosaics at a frequency greater than 0.1%. We are still examining two other lines and are exploring other means of inducing high-frequencies of X-linked mosaics . We have observed loss of X-chromosomes from worms irradiated by X- rays from 137Cs. The loss is seen in the F2 of irradiated worms (or the F1 or irradiated embryos). The number of males in this generation is in the range of 4-10%. The frequency of males is not distributed according to a Poisson but rather shows 'jack-pots' as might be expected from events that hit individual X chromosomes. The frequency of males in a jackpot can be as high as 40%. Most hermaphrodite siblings of these males are normal and show no increase in the frequency of male progeny. We asked whether only the irradiated chromosome was lost in an animal heterozygous for a marked irradiated X-chromosome and an unmarked unirradiated X-chromosome. Hermaphrodites marked with the X- linked marker, unc-78, were irradiated and then mated to N2 males. Wild type progeny were picked, cloned and scored for male offspring. Both wild type and unc-78 males were found at approximately equal frequency; apparently either chromosome can be lost.

Klass MR (1977) Worm Breeder's Gazette "a method for isolating biochemical quantities of purified sperm."

Klass MR

[Worm Breeder's Gazette, 1977]

This procedure starts with a predominantly male culture isolated by the Nitex filter method described previously. I usually start with a culture 60-70% males (a total of 1 - 5 x 10+E5 males). These males are washed extensively with 4 liters of M-9 salts over a 20 m Nitex filter to remove the larvae (especially the L1 which can be a problem later!) and to remove the E. coli. The washed male culture is concentrated by settling into 2-3 ml of M-9 salts. The worms are then spread evenly in very small disperse droplets over the surface of an 11'x7'x3/8' Plexiglass plate. Another Plexiglass plate of equal size is placed on top. The worms are then squashed between these plates in a Carver Laboratory Press with a manual hydraulic jack. The worms are subjected to 15,000 psi. This pressure causes the males to eject their sperm as well as their intestinal cells. However the sperm are the only free floating cells. The intestinal cells of both the males and hermaphrodites adhere to the Plexiglass. The plates are then pried apart and rinsed off. The rinse is collected in a glass trough and placed in 125 ml conical centrifuge tubes on ice. Everything is kept on ice from this point on. (If some males don't get squashed in the first run, they can be squashed again, although resquashing usually only gives 10% additional sperm.) The rinse fluid contains the sperm (and worm carcasses) and is filtered through two 10 m Nitex filters fitted to a 25 mm millipore filter holder of the type used on syringes. This removes the worm carcasses and larger debris. The filtrate is centrifuged in a clinical centrifuge on setting 6 for five minutes. The supernatant is discarded and the pelleted sperm are washed in M-9 salts and filtered through an 8 m polycarbonate Nucleopore filter fitted to a 25 mm millipore filter holder. I routinely obtain 10+E7-10+E8 sperm, an average of about 100 sperm per male. One dimensional SDS polyacrylamide gel patterns of these sperm closely match the patterns seen on autoradiographs of hermaphrodites mated with radioactively labeled males. Some additional information: Sperm can be stored overnight in M-9 salts with a minimum of lysis (1%) but they seem more fragile and after 48 hours show 50% lysis. I have found that sonication works best to lyse sperm rather than freeze/thawing. I have used M-9 salts throughout these procedures and have not tried Ascaris Ringer's solution. These purified sperm are currently being used to investigate cell surface proteins, DNA binding proteins, sperm actin and other sperm- specific proteins and their modifications in various spermatogenesis mutants as well as being used as a source of germ line DNA.