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Mammals in arid zones have to trade off thermal stress, predation pressure, and time spent foraging in a complex thermal landscape. We quantified the relationship between the environmental heat load and activity of a mammal community in the hot, arid Kalahari Desert. We deployed miniature black globe thermometers within the existing Snapshot Safari camera trap grid on Tswalu Kalahari Reserve, South Africa. Using the camera traps to record species' activity throughout the 24-h cycle, we quantified changes in the activity patterns of mammal species in relation to heat loads in their local environment. We compared the heat load during which species were active between two sites with differing predator guilds, one where lion ( Panthera leo ) biomass dominated the carnivore guild and the other where lions were absent. In the presence of lion, prey species were generally active under significantly higher heat loads, especially during the hot and dry spring. We suggest that increased foraging under high heat loads highlights the need to meet nutritional requirements while avoiding nocturnal activity when predatory pressures are high. Such a trade-off may become increasingly costly under the hotter and drier conditions predicted to become more prevalent as a result of climate change within the arid and semi-arid regions of southern Africa., Competing Interests: None., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The accuracy of predictive models (also known as mechanistic or causal models) of animal responses to climate change depends on properly incorporating the principles of heat transfer and thermoregulation into those models. Regrettably, proper incorporation of these principles is not always evident. We have revisited the relevant principles of thermal physiology and analysed how they have been applied in predictive models of large mammals, which are particularly vulnerable, to climate change. We considered dry heat exchange, evaporative heat transfer, the thermoneutral zone and homeothermy, and we examined the roles of size and shape in the thermal physiology of large mammals. We report on the following misconceptions in influential predictive models: underestimation of the role of radiant heat transfer, misassignment of the role and misunderstanding of the sustainability of evaporative cooling, misinterpretation of the thermoneutral zone as a zone of thermal tolerance or as a zone of sustainable energetics, confusion of upper critical temperature and critical thermal maximum, overestimation of the metabolic energy cost of evaporative cooling, failure to appreciate that the current advantages of size and shape will become disadvantageous as climate change advances, misassumptions about skin temperature and, lastly, misconceptions about the relationship between body core temperature and its variability with body mass in large mammals. Not all misconceptions invalidate the models, but we believe that preventing inappropriate assumptions from propagating will improve model accuracy, especially as models progress beyond their current typically static format to include genetic and epigenetic adaptation that can result in phenotypic plasticity., (© 2018 The Authors. Journal of Animal Ecology © 2018 British Ecological Society.)

Some mammals have the ability to lower their hypothalamic temperature below that of carotid arterial blood temperature, a process termed selective brain cooling. Although the requisite anatomical structure that facilitates this physiological process, the carotid rete, is present in members of the Cetartiodactyla, Felidae and Canidae, the carotid rete is particularly well developed in the artiodactyls, e.g. antelopes, cattle, sheep and goats. First described in the domestic cat, the seemingly obvious function initially attributed to selective brain cooling was that of protecting the brain from thermal damage. However, hyperthermia is not a prerequisite for selective brain cooling, and selective brain cooling can be exhibited at all times of the day, even when carotid arterial blood temperature is relatively low. More recently, it has been shown that selective brain cooling functions primarily as a water-conservation mechanism, allowing artiodactyls to save more than half of their daily water requirements. Here, we argue that the evolutionary success of the artiodactyls may, in part, be attributed to the evolution of the carotid rete and the resulting ability to conserve body water during past environmental conditions, and we suggest that this group of mammals may therefore have a selective advantage in the hotter and drier conditions associated with current anthropogenic climate change. A better understanding of how selective brain cooling provides physiological plasticity to mammals in changing environments will improve our ability to predict their responses and to implement appropriate conservation measures.

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access., Competing Interests: WMS, CC, JS, EK, BV, JR, MMS, and PWD are employees of Cynvenio Biosystems. WMS, BV, and PWD are inventors on issued patents or patent applications associated with this work and owned by Cynvenio Biosystems.

The use of selective brain cooling, where warm arterial blood destined for the brain is cooled in the carotid rete via counter-current heat exchange when in close proximity to cooler venous blood, contributes to the conservation of body water. We simultaneously measured carotid blood and hypothalamic temperature in four gemsbok, five red hartebeest and six blue wildebeest to assess the extent to which these free-living animals, with varying water dependency, routinely rely on selective brain cooling. We investigated the hypothesis that innate differences in selective brain cooling exist in large, sympatric artiodactyls with varying water dependency. All three species used selective brain cooling, without any discernible differences in three selective brain cooling indices. GLMMs revealed no species differences in the threshold temperature for selective brain cooling (z = 0.79, P = 0.43), the magnitude (z = -0.51, P = 0.61), or the frequency of selective brain cooling use (z = -0.47, P = 0.64), after controlling for carotid blood temperature and black globe temperature. Comparison of anatomical attributes of the carotid retes of the three species revealed that the volume (F 2,9 = 5.54, P = 0.03) and height (F 2,9 = 5.43, P = 0.03) of the carotid rete, per kilogram body mass, were greater in the red hartebeest than in the blue wildebeest. Nevertheless, intraspecific variability in the magnitude, the frequency of use, and the threshold temperature for selective brain cooling exceeded any interspecific variability in the three indices of selective brain cooling. We conclude that the three species have similar underlying ability to make use of selective brain cooling in an environment with freely available water. It remains to be seen to what extent these three species would rely on selective brain cooling, as a water conservation mechanism, when challenged by aridity, a condition likely to become prevalent throughout much of southern Africa under future climate change scenarios.

In artiodactyls, arterial blood destined for the brain can be cooled through counter-current heat exchange within the cavernous sinus via a process called selective brain cooling. We test the hypothesis that selective brain cooling, which results in lowered hypothalamic temperature, contributes to water conservation in sheep. Nine Dorper sheep, instrumented to provide measurements of carotid blood and brain temperature, were dosed with deuterium oxide (D2O), exposed to heat for 8 days (40 ◦C for 6-h per day) and deprived of water for the last five days (days 3 to 8). Plasma osmolality increased and the body water fraction decreased over the five days of water deprivation, with the sheep losing 16.7% of their body mass. Following water deprivation, both the mean 24h carotid blood temperature and the mean 24h brain temperature increased, but carotid blood temperature increased more than did brain temperature resulting in increased selective brain cooling. There was considerable inter-individual variation in the degree to which individual sheep used selective brain cooling. In general, sheep spent more time using selective brain cooling, and it was of greater magnitude, when dehydrated compared to when they were euhydrated. We found a significant positive correlation between selective brain cooling magnitude and osmolality (an index of hydration state). Both the magnitude of selective brain cooling and the proportion of time that sheep spent selective brain cooling were negatively correlated with water turnover. Sheep that used selective brain cooling more frequently, and with greater magnitude, lost less water than did conspecifics using selective brain cooling less efficiently. Our results show that a 50 kg sheep can save 2.6L of water per day (~60% of daily water intake) when it employs selective brain cooling for 50% of the day during heat exposure. We conclude that selective brain cooling has a water conservation function in artiodactyls.

Background: Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available., Methodology/principal Findings: Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL., Conclusions/significance: The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.

Predicting whether behaviour could buffer the effects of climate change on long-lived mammals requires a better understanding of the long-term behavioural responses of mammals to environmental stress. Using biologging, we measured locomotor activity and microclimate selection, over eight months, in five Arabian oryx (Oryx leucoryx) living free in a Saudi Arabian desert. The oryx displayed seasonal flexibility in activity patterns, shifting from a continuous 24-h activity pattern with crepuscular peaks in cooler months to a predominantly nocturnal activity pattern during the hottest months, without reducing the total 24-h activity level. The proportion of total 24-h activity that occurred during daylight hours was just 29±8% during the hottest months, versus 53±8% (mean±SD, n=5 oryx) in the other months. The attenuation in diurnal activity levels during the hot months was accompanied by the selection of cooler microclimates, presumably via shade seeking, during the heat of the day. Analysis of miniature black globe (miniglobe) temperature from a remote sensor on the collar of two female animals revealed that oryx selected microclimates cooler than the microclimates in direct sun at higher environmental heat loads across all periods, but with enhanced efficiency during the dry periods. We have quantified activity re-assignment and microclimate selection as responses to hot arid conditions in a free-living artiodactyl. Such flexible behavioural processes may act to buffer the adverse effects of the progressively hotter and drier conditions predicted to occur with climate change., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Selective brain cooling is a thermoregulatory effector proposed to conserve body water and, as such, may help artiodactyls cope with aridity. We measured brain and carotid blood temperature, using implanted data loggers, in five Arabian oryx (Oryx leucoryx) in the desert of Saudi Arabia. On average, brain temperature was 0.24±0.05°C lower than carotid blood temperature for four oryx in April. Selective brain cooling was enhanced in our Arabian oryx compared with another species from the same genus (gemsbok Oryx gazella gazella) exposed to similar ambient temperatures but less aridity. Arabian oryx displayed a lower threshold (37.8±0.1°C vs 39.8±0.4°C), a higher frequency (87±6% vs 15±15%) and a higher maximum magnitude (1.2±0.2°C vs 0.5±0.3°C) of selective brain cooling than did gemsbok. The dominant male oryx displayed less selective brain cooling than did any of the other oryx, but selective brain cooling was enhanced in this oryx as conditions became hotter and drier. Enhanced selective brain cooling in Arabian oryx supports the hypothesis that selective brain cooling would bestow survival advantages for artiodactyl species inhabiting hot hyper-arid environments.

Heterothermy, a variability in body temperature beyond the normal limits of homeothermy, is widely viewed as a key adaptation of arid-adapted ungulates. However, desert ungulates with a small body mass, i.e. a relatively large surface area-to-volume ratio and a small thermal inertia, are theoretically less likely to employ adaptive heterothermy than are larger ungulates. We measured body temperature and activity patterns, using implanted data loggers, in free-ranging Arabian oryx (Oryx leucoryx, ±70 kg) and the smaller Arabian sand gazelle (Gazella subgutturosa marica, ±15 kg) inhabiting the same Arabian desert environment, at the same time. Compared to oryx, sand gazelle had higher mean daily body temperatures (F(1,6) = 47.3, P = 0.0005), higher minimum daily body temperatures (F(1,6) = 42.6, P = 0.0006) and higher maximum daily body temperatures (F(1,6) = 11.0, P = 0.02). Despite these differences, both species responded similarly to changes in environmental conditions. As predicted for adaptive heterothermy, maximum daily body temperature increased (F(1,6) = 84.0, P < 0.0001), minimum daily body temperature decreased (F(1,6) = 92.2, P < 0.0001), and daily body temperature amplitude increased (F(1,6) = 97.6, P < 0.0001) as conditions got progressively hotter and drier. There were no species differences in activity levels, however, both gazelle and oryx showed a biphasic or crepuscular rhythm during the warm wet season but shifted to a more nocturnal rhythm during the hot dry season. Activity was attenuated during the heat of the day at times when both species selected cool microclimates. These two species of Arabian ungulates employ heterothermy, cathemerality and shade seeking very similarly to survive the extreme, arid conditions of Arabian deserts, despite their size difference.

Heterothermy, a variability in body temperature beyond the limits of homeothermy, has been advanced as a key adaptation of Arabian oryx (Oryx leucoryx) to their arid-zone life. We measured body temperature using implanted data loggers, for a 1-year period, in five oryx free-living in the deserts of Saudi Arabia. As predicted for adaptive heterothermy, during hot months compared to cooler months, not only were maximum daily body temperatures higher (41.1 ± 0.3 vs. 39.7 ± 0.1°C, P = 0.0002) but minimum daily body temperatures also were lower (36.1 ± 0.3 vs. 36.8 ± 0.2°C, P = 0.04), resulting in a larger daily amplitude of the body temperature rhythm (5.0 ± 0.5 vs. 2.9 ± 0.2°C, P = 0.0007), while mean daily body temperature rose by only 0.4°C. The maximum daily amplitude of the body temperature rhythm reached 7.7°C for two of our oryx during the hot-dry period, the largest amplitude ever recorded for a large mammal. Body temperature variability was influenced not only by ambient temperature but also water availability, with oryx displaying larger daily amplitudes of the body temperature rhythm during warm-dry months compared to warm-wet months (3.6 ± 0.6 vs. 2.3 ± 0.3°C, P = 0.005), even though ambient temperatures were the same. Free-living Arabian oryx therefore employ heterothermy greater than that recorded in any other large mammal, but water limitation, rather than high ambient temperature, seems to be the primary driver of this heterothermy.

Defining the identity of embryonic stem (ES) cells in quantitative molecular terms is a prerequisite to understanding their functional characteristics. Little is known about the role of microRNAs (miRNAs) in the regulation of ES cell identity. Statistical analysis of miRNA expression revealed unique expression signatures that could definitively classify mouse ES (mES), embryoid bodies (mEB), and somatic tissues. Analysis of these data sets also provides further confirmation of the nonrestrictive expression of miRNAs during murine development. Using combined genome-wide expression analyses of both miRNAs and mRNAs, we observed both negative and positive correlations in gene expression between miRNAs and their predicted targets. ES-specific miRNAs were positively correlated with their predicted targets, suggesting that mES-specific miRNAs may have a different role or mechanism in regulating their targets in mES maintenance or differentiation. The concept of cellular identity has changed with technology; this study redefines cellular identity by a generic statistical method of known dimension.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate mRNAs through a sequence-specific mechanism. By virtue of their structure and mechanism of action, computational methods have been devised to investigate the encoding of miRNA genes and the targets of miRNA action. A variety of assumptions have predicated the implementation of these various computational solutions. Evolutionary sequence conservation, secondary structure, and folding energetics are some of the assumptions that have been used. The success of these different computational solutions has been evaluated for both elucidation of new miRNAs and deducing targets of miRNA action. While the focus is on search techniques for new miRNAs, we have compared the programs miRseeker, miRScan, PalGrade, ProMiR, and miRAlign as examples of implementation of these techniques. For these programs, a benchmark comparison between theoretical estimation and actual identification is possible. We have also compared the target prediction programs TargetScanS, PicTar, DIANA-microT, miRanda, and RNAhybrid. However, it is difficult to rigorously assess the benchmark performance of these programs due to the difficulty in confirming their theoretical predictions.

During the last decade, a variety of critical biological processes, including early embryo development, cell proliferation, differentiation, apoptosis, and metabolic regularity, have been shown to be genetically regulated by a large gene family encoding a class of tiny RNA molecules termed microRNAs (miRNAs). All miRNAs share a common biosynthetic pathway and reaction mechanisms. The sequence of many miRNAs is found to be conserved, in their mature form, among different organisms. In addition, the evolutionary appearance of multicellular organisms appears to correlate with the appearance of the miRNA pathway for regulating gene expression. The miRNA pathway has the potential to regulate vast networks of gene products in a coordinate manner. Recent evidence has not only implicated the miRNA pathway in regulating a vast array of basic cellular processes but also specialized processes that are required for cellular identity and tissue specificity. A survey of the literature shows that some miRNA pathways are conserved virtually intact throughout phylogeny while miRNA diversity also correlates with speciation. The number of miRNA genes, the expression of miRNAs, and target diversities of miRNAs tend to be positively correlated with morphological complexities observed in animals. Thus, organismal complexity can be estimated by the complexity of the miRNA circuitry. The complexity of the miRNA gene families establishes a link between genotypic complexity and phenotypic complexity in animal evolution. In this paper, we start with the discussion of miRNA conservation. Then we interpret the trends in miRNA conservation to deduce miRNA evolutionary trends in metazoans. Based on these conservation patterns observed in each component of the miRNA regulatory system, we attempt to propose a global insight on the probable consistency between morphological evolution in animals and the molecular evolution of miRNA gene activity in the cell.

During different periods of mammalian development, global changes in gene expression occur. Developmental changes in global gene expression have been modeled as a restrictive process. To test the restriction model of global changes in gene expression, we have used embryonic stem (ES) cells as a model system for the early mammalian embryo. ES cells are pluripotent cells that can contribute to all cellular lineages of the developing mammalian fetus and are derived from early embryonic cells. Using this model system, we have studied a new class of RNAs called microRNAs that have been identified and shown to play a role in the direct regulation of messenger RNAs. Here we report the expression signature for 248 microRNAs in 13 independent murine ES cells, embryoid bodies, and somatic tissues. The expression profile for 248 mouse microRNAs was determined for embryonic stem cells, embryoid bodies, mouse embryos, mature heart, lung, liver, kidney, and brain. Characteristic microRNA expression signatures were observed for each evaluated sample. When the characteristic microRNA signatures for developmentally ordered samples were compared, immature samples exhibited a less complex microRNA transcript profile than did mature samples. Our data support a progressive model of microRNA gene expression. Based on the progressive increase in complexity of micro- RNA expression, we hypothesize that the mammalian developmental program requires a temporal coupling of expression between microRNAs and messenger RNAs to enable the developmental potential observed in mammalian ontogeny.

Development in mammals is a complex process requiring gene expression to be spatially and temporally well-regulated. Factors modulate gene functioning by controlling transcription, translation, or mRNA degradation. microRNAs (miRNAs) are a group of small RNA molecules (approximately 22 nucleotides) that attenuate gene activity posttranscriptionally by suppressing translation or destabilizing mRNAs. miRNAs have been recently validated to regulate many animal developmental events including proliferation, differentiation, and apoptosis. Many miRNAs display intriguing expression and functioning patterns throughout these pathways. Here we will review achievements to date about studies of how miRNAs affect a variety of animal developmental transitions, from the formation of early embryos to the generation of highly specialized tissues., (Copyright 2006 Wiley-Liss, Inc.)

The noncoding RNA Xist has been shown to direct the mammalian dosage compensation pathway. Expression of the Xist RNA is regulated through an uncharacterized post-transcriptional mechanism, thought to involve Xist RNA stability. We have previously demonstrated that Xist RNA isoforms contain different 3' ends. In this report we analyze the expression patterns of Xist RNA isoforms and show the Xist RNA long form (L-isoform) is the predominant form in early development. Significant amounts of both the short form (S-isoform) and the L-isoform were found in the female soma. We also define the precise sequence structure of the Xist RNA isoforms 3' ends and show the S-isoform and the L-isoform are structurally dissimilar. Our data show both the S-isoform and L-isoform are cleaved from the same primary transcript. However, the S-isoform is subsequently post-transcriptionally polyadenylated, while the L-isoform is not post-transcriptionally polyadenylated. Sequence organization of the L-isoform shows that there are at least five different nonadenylated L-isoforms in the female soma and only one in embryonic stem (ES) cells. This stem cell-and somatic cell-specific processing may suggest a role for Xist RNA processing in the regulation of Xist RNA expression.

In the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation.

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteination and recovery of DNA. The main differences between various approaches lie in the extent of deproteination and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.

This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial-digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species-specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian-cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC- and human-derived probes.

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.

All hybridization methods depend upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their Tm (melting temperature). A detailed procedure is described in which bacteriophage plaques or bacterial colonies bound to a filter membrane are detected by hybridization with a radioactive probe. Hybridization proceeds on prewet filters placed in a sealable plastic bag. After hybridization the filters are removed from the sealed bag, excess probe is washed off, and the filters are autoradiographed to identify the clones that have hybridized with the probe. An Alternate Protocol differs mainly in that formamide is not used in the hybridization solution.

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteination and recovery of DNA. The main differences between various approaches lie in the extent of deproteination and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells., (Copyright 2001 Academic Press.)

Murine Xist is an essential transcript for X chromosome inactivation (X inactivation). According to recently revised structure, Xist is at least 17.8 kb long. It consists of seven exons and there are two major transcripts in female somatic cells. In this study we further defined the molecular structures of the two isoforms, namely short (S) and long (L) forms by northern blot and RNAse protection assay (RPA). The following lines of evidences suggest that mouse Xist depends on differential polyadenylation, not alternative splicing, to generate the two RNA isoforms: (1) only one band was detectable with the northern probes spanning the 3' end of Xist. (2) RPA showed the 3' termini of both S and L forms, and there are putative polyadenylation signals and hairpin structures close to these ends. (3) Analyses by splice site prediction program did not show any evidence of splice motifs in the sequence of L form. (4) Alignments between Xist 3' end (ESTs) and genomic sequence support the absence of splicing event in the region. The newly revised structure of Xist isoforms may have different stability and roles in the process of X inactivation.

We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes directed against interspersed alpha-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant alpha-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program "whole genome" approach based on approximately 400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, approximately 240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome "bar coding". Potential application of PNA probes for investigating repeat structure and function is also discussed.

The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the 3' end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH) demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences in the newly defined region show overall sequence similarity with the 3' terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two sub-regions that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist. al. 1992). This gene, called XIST/Xist (X inactive specific transcript), shows several interesting features. First, both human and mouse XIST/Xist cDNA are unusually long, reportedly 16.5 kb and 17.8 kb, respectively (Brown et al. 1992; Hong et al. 1999). Second, the transcript does not seem to encode a protein, on the basis of the lack of a significant open reading frame, absence of the Xist RNA from polysomes, and localization of the transcript in the nucleus (Brockdorff et al. 1992; Brown et al. 1992). Third, the XIST/Xist RNA physically associates with, or 'coats,' the inactive X Chr (Brown et al. 1992; Clemson et al. 1996). Fourth, XIST/Xist transcripts can be observed as early as the four-cell stage, and upon the initiation of X-inactivation, the steady-state level of the transcript rises dramatically, apparently by stabilization of the RNA (Panning et al. 1997; Sheardown et al. 1997). Although the function of XIST/Xist is not known, deletion of the gene leads to failure of X-inactivation, and knock-out mice die around the gastrulation stage (Marahrens et al. 1997; Penny et al. 1996). In this report, we revise the structure of the human XIST cDNA and discuss structural features of the newly defined region.

In this report, we present structural data for the murine Xist gene. The data presented in this paper demonstrate that the murine Xist transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine Xist gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3' end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3' end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of Xist are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the Xist 3' end in the process of X-chromosome counting and choice during embryonic development.

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.

To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells. In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture.

X inactivation results in inactivation of one X chromosome to compensate for gene dosage differences between mammalian females and males. It requires the X-inactivation center (Xic) and Xist in cis. We report that introducing 450 kb of murine Xic/Xist sequences onto autosomes activates female dosage compensation in male ES cells. Xist is induced upon differentiation and can be expressed from both endogenous and ectopic loci, suggesting that elements for counting and choosing Xs are present in the transgene. Differentiating transgenic ES cells undergo excessive cell death. Postnatally, Xist is expressed only from the transgene. Ectopic Xist RNA structurally associates with the autosome and may inactivate a marker gene in cis. These results argue that the Xic is contained within 450 kb and that these sequences are sufficient for chromosome counting, choosing, and initiation of X inactivation.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed that allows the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene.

We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys+ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys+ Trp- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated.

The cloning of large contiguous segments of mammalian DNA in Saccharomyces cerevisiae has become possible with the advent of Yeast Artificial Chromosomes (YACs). We are interested in extending the technique of genetic complementation analysis to the molecular level through the introduction of YACs into mammalian cells and the mammalian germline. We report the successful introduction of homogeneous DNA derived from a 150 kbp YAC spanning the murine Col1a1 locus into murine fibroblasts carrying a mutation at this locus. The YAC DNA was fractionated by pulse field electrophoresis, condensed with polyamines, and introduced into mutant fibroblasts via DNA-lipid micelles. The DNA was integrated as a stable intact unit in 10% of the transfected clones conferring collagen RNA expression to the mutant cells.

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

The human T cell receptor gamma locus, including eleven variable-region, five joining-region, and two constant-region segments, is contained in 160 kilobases. During T cell somatic development these genes undergo rearrangement by deletion of the sequences separating the variable and joining regions. The molecular map of this locus was completely defined by deletion mapping and restriction mapping. Restriction fragments were resolved by standard agarose electrophoresis and field inversion electrophoresis. These studies demonstrate that the deletions in this locus, which occur during the formation of a functional T cell receptor gamma-chain gene, range from 50 to 145 kilobases in length. These studies also provide a structural basis for understanding the development of the gamma-chain peptide repertoire, and extends the potential of the emerging pulsed-field electrophoretic technology.

We have cloned and sequenced two homologous J regions of the T cell gamma gene and localized one 5' of each constant region. They have been numbered J gamma 1.1 and J gamma 2.1; other J regions have been renamed in a similar manner. Southern blot analysis suggests that there is comparatively more rearrangement to these J regions in thymocytes than peripheral blood T cells. We propose that either there is gamma-chain gene rearrangement first to J gamma 1.1 or 2.1 and then to J gamma 1.3 or 2.3, or there is selected cell death of thymic T cells bearing a J gamma 1.1 or 2.1 rearrangement.

Cerebral ring hemorrhages were observed in four species of monkeys with various forms of anemia. The lesions resembled those described in humans with a variety of diseases, including pernicious anemia. The pale centers of most lesions were surrounded by rings of erythrocytes. Also observed were petechiae, pale foci without hemorrhage, and glial nodules. Ultrastructural study revealed that the pale cores were foci of degenerating neurites associated with infiltration of fibrin into the finest interstitial spaces. Leakage of firbinogen and erythrocytes apparently occurred from capillary sized vessels. A few of these were thrombosed, some were normal and some had only small fibrin thrombi in apparent apposition to damaged endothelial tight junctions. These findings suggest that a primary effect of anemia and other insults associated with ring hemorrhages is damage to tight junctions. Consequent fibrinous exudation clogs the interstitium and results in neuritic degeneration.

Characterization of tumors that arise spontaneously in the AKR mouse indicates that they are derived from cells of a distinct T-cell lineage. Cells in this subclass bear surface antigens, designated Tpre, Tthy, Tind, and Tsu, which are encoded by genes in the Tsu linkage group on murine chromosome 12. We have examined the rearrangement and expression of genes encoding the T-cell alpha, beta, and gamma chains in these tumors. Although these cells contain alpha-chain mRNA, they do not produce a normal-sized beta-chain mRNA. Most of them also lack gamma-chain mRNA. Each thymic leukemia was derived from a cell arrested at a different stage of development as defined by their expression of terminal deoxynucleotidyl transferase and Thy-1 mRNA. The data presented here are consistent with a model in which thymocytes expressing Tpre, Tthy, Tind, or Tsu undergo somatic development parallel to the development of other T cells. However, these thymocytes do not appear to differentiate into cells bearing alpha-beta heterodimers of the T-cell antigen receptor.

This paper presents a brief review of the history of the use of conductance and impedance techniques for monitoring bacterial growth and activity. The paper also describes the development of an impedance methodology applicable to the estimation of numbers of total coliform bacteria in domestic sewage and other wastewaters.

The human T cell receptor gamma chain locus encodes the immunoglobulin-like gamma chain polypeptide and spans a distance of approximately 150 kb. Previous studies have not precisely characterized the interval separating variable regions from joining--constant regions which is excised during gamma gene rearrangement. We report a series of overlapping cosmids which includes the portion of the gamma chain locus beginning with V2 and extends to the second exon of C2. Sixteen kilobases separate the most 3' variable region gene, V4, from the most 5' joining segment, J1.1.