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Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This... There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes). MLH1 foci correspond to the location of recombination nodules (RNs) that mark sites of crossing over. We found that MLH1 foci occur at similar frequencies per unit length of SC in interstitial knobs and in the 1 µm segments of SC in euchromatin immediately to either side of interstitial knobs. These results indicate not only that crossing over occurs within knob heterochromatin, but also that crossing over is not suppressed in the context of SC length in maize knobs. However, because there is more DNA per unit length of SC in knobs compared to euchromatin, crossing over is suppressed (but not eliminated) in knobs in the context of DNA length compared to adjacent euchromatin.

Eggs of domestic fowl were given daily injections of vehicle (DMSO) or vehicle plus acetazolamide, a potent inhibitor of the enzyme carbonic anhydrase, beginning on day 12 of incubation. Embryos were removed from eggs on days 16 and 18, and carcasses and yolks were analyzed for calcium, magnesium, and phosphorus. Treatment with acetazolamide did not affect the quantity of calcium or phosphorus in carcasses and the effect, if any, on magnesium in carcasses was small. However, calcium content of yolk was reduced substantially by acetazolamide both on day 16 and day 18. The reduction in calcium content of yolk led, in turn, to a reduction in the total quantity of calcium in eggs on days 16 and 18. Embryos exposed to acetazolamide seemingly mobilized less calcium from the eggshell than did control embryos. When faced with a shortfall in the availability of calcium from the eggshell, embryos defended carcass calcium, and the shortfall was reflected in a reduction in the quantity of calcium deposited in yolk. The results of this study support the concept that the enzyme carbonic anhydrase plays a role in solubilization of the eggshell and provision of calcium to embryos.

We assessed the ability of hatchling painted turtles (Chrysemys picta) from northern Minnesota to resist freezing when exposed to conditions like those occurring in natural hibernacula (natal nests). We placed animals individually into artificial hibernacula constructed in jars of damp, loamy sand and then lowered the temperature to approximately -0.4°C, which was below the equilibrium freezing point for water in the soil (approximately -0.1°C) but above that for body fluids of the turtles (approximately -0.7°C). Next, ice was used to initiate freezing of water in the soil, after which the substratum was allowed to freeze to an equilibrium temperature of -0.4°C. The minimum temperature then was reduced by 1°C/day to either -2.5 or -6.5°C. The minimum was maintained for 9 days in the former treatment and for 5 days in the latter, so that turtles in both treatments were exposed for the same length of time to temperatures below the equilibrium freezing point for their body fluids. Some animals in each treatment were inoculated by ice crystals that penetrated their integument, and these turtles froze and died. However, many other animals resisted inoculation, remained unfrozen, and survived the test of tolerance. We conclude that hatchling painted turtles from northern Minnesota, like those from North Dakota, north-central Nebraska, and northern Illinois, withstand the cold of midwinter by supercooling and not by tolerating freezing.

International audience; Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, we combine the efficiency of light microscopy for revealing cellular functions using fluorescent probes with the high resolution of electron microscopy to localize and characterize COs in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I and II COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.

We monitored temperatures during the winter of 1995–1996 inside 18 nests containing hatchling painted turtles (Chrysemys picta). The study was performed at the Valentine National Wildlife Refuge in north-central Nebraska to assess survival of neonatal turtles in relation to the thermal environment inside their hibernacula. Minimum temperatures in the nests varied from −3 to −21 °C, and were better predictors of survival of hatchlings than other measures of the thermal environment. All hatchlings survived in nests where the temperature never went below −7 °C, some animals survived in nests where the minimum was between −7 and −13 °C, but no turtle survived in a nest where the minimum was below −14 °C. Hatchlings probably survived the cold by sustaining a state of supercooling, because the duration of exposure to low temperatures was far too long for animals in most nests to have survived in a frozen state.

Proteins of the cohesin complex are essential for sister chromatid cohesion and proper chromosome segregation during both mitosis and meiosis. Cohesin proteins are also components of axial elements/lateral elements (AE/LEs) of synaptonemal complexes (SCs) during meiosis, and cohesins are thought to play an important role in meiotic chromosome morphogenesis and recombination. Here, we have examined the cytological behavior of four cohesin proteins (SMC1, SMC3, SCC3, and REC8/SYN1) during early prophase I in tomato microsporocytes using immunolabeling. All four cohesins are discontinuously distributed along the length of AE/LEs from leptotene through early diplotene. Based on current models for the cohesin complex, the four cohesin proteins should be present at the same time and place in equivalent amounts. However, we observed that cohesins often do not colocalize at the same AE/LE positions, and cohesins differ in when they load onto and dissociate from AE/LEs of early prophase I chromosomes. Cohesin labeling of LEs from pachytene nuclei is similar through euchromatin, pericentric heterochromatin, and kinetochores but is distinctly reduced through the nucleolar organizer region of chromosome 2. These results indicate that the four cohesin proteins may form different complexes and/or perform additional functions during meiosis in plants, which are distinct from their essential function in sister chromatid cohesion.

We studied tolerance for cold in hatchling painted turtles (Chrysemys picta) from Lake Metigoshe, Bottineau County, North Dakota, to determine whether neonates in populations near the northern limit of distribution rely on a tolerance for freezing or on a capacity for supercooling to survive their first winter of life. We placed hatchlings individually into artificial hibernacula constructed in jars of damp, loamy sand and then cooled the jars to approximately -0.45 degrees C, which was below the equilibrium freezing point for water held by the sand but above that for body fluids of the neonatal turtles. A piece of ice next was placed on the surface of the sand in each jar to induce freezing of the soil water. After the soil water had frozen to an equilibrium, the temperature in the jars was lowered by 1 degrees C/d to minima averaging -2.5 degrees C, -4.5 degrees C, -6.5 degrees C, and -10.5 degrees C in different treatments. These temperatures were maintained for varying periods, so that animals in each treatment were exposed to temperatures below the equilibrium freezing point for their body fluids for a total of 11 d. Thirty of 32 hatchlings survived exposure to -2.5 degrees C; 24 of 32 survived at -4.5 degrees C; 14 of 32 withstood -6.5 degrees C; and 7 of 32 tolerated -10.5 degrees C. Freezing exotherms were detected in temperature profiles for turtles that succumbed but not in those for hatchlings that survived. Thus, the ability of hatchlings to withstand subzero temperatures for extended periods apparently requires that they avoid freezing. Although other workers contend that tolerance for freezing is the key to survival over winter by hatchling painted turtles from the region of Lake Metigoshe, our findings indicate that neonates rely primarily on their ability to remain unfrozen and supercooled.

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