Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.     

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.     

A mobile cage facilitates periodic social contact and exercise for singly caged adult Vervet monkeys

Abstract: A mobile exercise cage that expands the quantity and improves the quality of the space available to singly caged adult Vervet monkey males is described. It was easily fitted into an existing caging system and the addition of a resident consort female made it possible for the males to mate and have regular social contact.     

Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation.

Activation of telomerase in human cancers is thought to be necessary to overcome the progressive loss of telomeric DNA that accompanies proliferation of normal somatic cells. According to this model, telomerase provides a growth advantage to cells in which extensive terminal sequence loss threatens viability. To test these ideas, we have examined telomere dynamics and telomerase activation during mammary tumorigenesis in mice carrying a mouse mammary tumor virus long terminal repeat-driven Wnt-1 transgene. We also analyzed Wnt-1-induced mammary tumors in mice lacking p53 function. Normal mammary glands, hyperplastic mammary glands, and mammary carcinomas all had the long telomeres (20 to 50 kb) typical of Mus musculus and did not show telomere shortening during tumor development. Nevertheless, telomerase activity and the RNA component of the enzyme were consistently upregulated in Wnt-1-induced mammary tumors compared with normal and hyperplastic tissues. The upregulation of telomerase activity and RNA also occurred during tumorigenesis in p53-deficient mice. The expression of telomerase RNA correlated strongly with histone H4 mRNA in all normal tissues and tumors, indicating that the RNA component of telomerase is regulated with cell proliferation. Telomerase activity in the tumors was elevated to a greater extent than telomerase RNA, implying that the enzymatic activity of telomerase is regulated at additional levels. Our data suggest that the mechanism of telomerase activation in mouse mammary tumors is not linked to global loss of telomere function but involves multiple regulatory events including upregulation of telomerase RNA in proliferating cells.     

Selected locus and multiple panel models for radiation hybrid mapping.

We develop two new types of models for whole-genome radiation hybrid mapping using the general multipoint framework. The first, selected locus models, are appropriate for mapping markers in the region of a selectable locus that was used in creation of the hybrids. The models allow for strong retention of the selectable locus, with retention rates decreasing with increasing distance from the selectable locus in both directions. We illustrate the application of these models with 10 chromosome 17 sequence-tagged site (STS) markers and the thymidine kinase (TK) locus typed on a whole-genome hybrid panel in which TK was used in the selection process. The second set of models are appropriate when loci typed on two or more independent panels are to be used to build maps. Maps can be built assuming interlocus distances are independent or proportional between the panels, and the hypothesis of proportional distances can be tested. We illustrate the application of these models by using 27 chromosome 21 STS markers typed on two hybrid panels created with radiation doses of approximately 10,000 and approximately 50,000 Rads.     

Unusual chromatin in human telomeres.

We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.     

Carotenoid composition and metabolism in green and blue-green algal lichens in the field

The carotenoid composition of 33 species of green algal lichens and 5 species of blue-green algal lichens was examined and compared with that of the leaves of higher plants. As in higher plants, green algal lichen species which were found in both shade and full sunlight exhibited higher levels of the carotenoids involved in photoprotective thermal energy dissipation (zeaxanthin as well as the total xanthophyll cycle pool) in the sun than in the shade. This was particularly true when thalli were moist during exposure to high light, or presumably became desiccated in full sunlight. However, the reverse trend in the carotenoid composition of green algal lichens was also observed in those species which were found predominantly either in the shade or in full sunlight. In this case sun-exposed lichens often possessed lower levels of zeaxanthin and of the components of the xanthophyll cycle than lichens which were found in the shade. In contrast to higher plants, the lichens from all habitats exhibited a relatively high ratio of carotenoids to chlorophylls (more characteristic of sun leaves), very low levels of α-carotene (similar to that found in sun leaves), and a level of β-carotene similar to that found in shade leaves. Zeaxanthin, but not the expoxides of the xanthophyll cycle, was also frequently found in blue-green algal lichens. A trend for increasing levels of zeaxanthin with increasing growth light regime was observed inPeltigera rufescens, the species which was found to occur over the widest range of light environments. The level of zeaxanthin per chlorophylla in these blue-green algal lichens was in a range similar to that per chlorophylla+b in green algal lichens. However, zeaxanthin was also absent in one species,Collema cristatum, in full sunlight. Thus, the zeaxanthin content of the blue-green algal lichens can be similar to that of higher plants, or it can be rather dissimilar, as was also the case in the green algal lichen species. The presence of large amounts of ketocarotenoids in blue-green algal lichens is also noteworthy.     

Temperate rainforest lichens in New Zealand: high thallus water content can severely limit photosynthetic CO2 exchange

CO₂ exchange rate in relation to thallus water content (WC, % of dry weight) was determined for 22 species of lichens, mainly members of the genera Pseudocyphellaria and Sticta, from a temperate rainforest, Urewere National Park, New Zealand. All data were obtained in the field, either using a standard technique in which the lichens were initially wetted (soaked or sprayed, then shaken) and allowed to slowly dry, or from periodic measurements on samples that were continuously exposed in their natural habitat. A wide range of WC was found, with species varying from 357 to 3360% for maximal WC in the field, and from 86 to 1300% for optimal WC for photosynthesis. Maximal WC for lichens, wetted by the standard technique, were almost always much less than the field maxima, due to the presence of water on the thalli. The relationships between CO₂ exchange rate and WC could be divided into four response types based on the presence, and degree, of depression of photosynthesis at high WC. Type A lichens showed no depression, and Type B only a little at maximal WC. Type C had a very large depression and, at the highest WC, CO₂ release could occur even in the light. Photosynthetic depression commenced soon after optimal WC was reached. Type D lichens showed a similar depression but the response curve had an inflection so that net photosynthesis was low but almost constant, and never negative, at higher WC. There was little apparent relationship between lichen genus or photobiont type and the response type. It was shown that high WC does limit photosynthetic CO₂ uptake under natural conditions. Lichens, taken directly from the field and allowed to dry under controlled conditions, had net photosynthesis rates that were initially strongly inhibited but rose to an optimum, before declining at low WC. The limiting effects of high WC were clearly shown when, under similar light conditions, severe photosynthetic depression followed a brief, midday, rain storm. Over the whole measuring period the lichens were rarely at their optimal WC for photosynthesis, being mostly too wet or, occasionally, too dry. Photosynthetic performance by the lichens exposed in the field was similar to that expected from the relationship between the photosynthetic rate and WC established by the standard procedure.     

Match probabilities in racially admixed populations.

The calculation of match probabilities is the most contentious issue dividing prosecution and defense experts in the forensic applications of DNA fingerprinting. In particular, defense experts question the applicability of the population genetic laws of Hardy-Weinberg and linkage equilibrium to racially admixed American populations. Linkage equilibrium justifies the product rule for computing match probabilities across loci. The present paper suggests a method of bounding match probabilities that depends on modeling gene descent from ancestral populations to contemporary populations under the assumptions of Hardy-Weinberg and linkage equilibrium only in the ancestral populations. Although these bounds are conservative from the defendant's perspective, they should be small enough in practice to satisfy prosecutors.