Expression of β-glucuronidase haplotypes in prototype and congenic mouse strains

A gene complex consists of a structural gene with its associated regulatory information; together they behave as the functional and evolutionary unit of mammalian chromosomes. The use of congenic lines, in which alternate forms, or haplotypes, of a gene complex are transferred into a common genetic background by repeated backcrossing, provides a means of comparing the regulatory properties of different haplotypes of a gene complex without the complications introduced by extraneous genetic differences. We have now carried out such a study of the A, B, and H haplotypes of the -glucuronidase gene complex, [Gus], in mice. These haplotypes were derived from strains A/J, C57BL/6J, and C3H/HeJ and were compared against the C57BL/6J genetic background. Enzyme structure was compared in terms of charge (isoelectric point), stability (rate of thermal denaturation), substrate affinity (for 4 MU glucuronide), and antigenicity (reactivity with a standard antibody). Compared to the B form, the enzyme coded by the A haplotype has a lower isoelectric point, and that coded by the H haplotype is less stable. The decreased stability is the result of a lower activation energy for the thermal denaturation reaction. These differences were maintained in the congenic strains. All three enzyme forms showed identical substrate affinities. Antigenicity per enzyme unit was also identical for all three, indicating that none lacks an antigenic site possessed by the others and that they all possess the same catalytic activity per molecule. The expression of alleles of the Gus-t temporal locus within the gene complex was not affected by transfer into the C57BL/6 genetic background. The same developmental switches in enzyme activity were seen in each case. Transfer into the C57Bl/6 background also did not affect expression of the Gus-r regulator determining androgen inducibility of -glucuronidase synthesis in kidney epithelial cells. However, enzyme accumulation in induced cells was altered when the haplotypes were transferred into the C57BL/6 genetic background. Since the rate of synthesis was not affected, it suggests that the genetic differences between strains that are not linked to the [Gus] complex affect the rate of enzyme loss by degradation or secretion. -Glucuronidase in liver is present in both lysosomes and endoplasmic reticulum (microsomes). The relative amount of enzyme at each site depended on both the indentity of the structural allele and the function of unlinked genetic modifiers. Within the C57BL/6 background the percentage of total enzyme present in the microsome fraction was the order A>B>H. For the H form of the enzyme the percentage was appreciably greater in the C3H genetic background compared to C57BL/6. As expected, then, the [Gus] complex contains all of the genetic determinants of enzyme structure detected by thermal stability and isoelectric point measurements. Additionally, the complex contains all of the genetically determined differences between strains in the regulation of -glucuronidase synthesis, including the programming of synthesis during development and the responsiveness of the [Gus] complex to hormonal stimulation. In contrast, genetic determinants of posttranslational processing are located elsewhere, including factors affecting enzyme localization and secretion/degradation. These results illustrate the utility of congenic strains for minimizing other genetic variables in characterizing the regulatory properties of alternate haplotypes of a gene complex.This work was supported by USPHS Research Grant GM 19521.     

Cross-species genetic markers: a useful tool to study the world's most threatened wild equid—Equus africanus

Once a diverse family, the Equidae family is now reduced to a single genus, Equus. From the seven extant species of the genus, the African wild ass (Equus africanus) is the most threatened with extinction (last survey indicated 600 individuals). In this work we tested 25 published microsatellite primer pairs isolated from the horse genome on 22 African wild ass (E. africanus) individuals from wildlife reserves and zoos. From the 25 loci tested, 15 amplified well and showed moderate allelic richness (5.06, mean number of alleles) and moderately high expected heterozigosity (0.59). Although all possible loci pairs showed no significant gametic disequilibrium (P?>?0.007), deviations from Hardy–Weinberg proportions were found in 2 out of the 15 analysed microsatellite loci (AHT5 and VHL20). Here, we propose these polymorphic markers to be used as a standard set in future studies on population and conservation genetics of the African wild ass.     

Synechocystis sp. PCC 6803 — a useful tool in the study of the genetics of cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 was the first phototrophic organism to be fully sequenced. The genomic sequence has revealed the structure of the genome and its gene constituents (3167 genes), as well as the relative map positions of each gene. The functions of nearly half of the genes has been deduced using similarity searches. The genome sequence has also allowed for the implementation of systematic strategies to study gene function and the mechanisms of gene regulation on a genome-wide level. Two genome databases, CyanoBase and CyanoMutants, have been established and act as a central repository for information on gene structure and gene function, respectively. As a result of the genome sequencing and the establishment of these databases, Synechocystis sp. PCC 6803 provides an extremely versatile and easy model to study the genetic systems of photosynthetic organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

The 1×C3 panel of recombinant inbred mouse strains. Presence or absence of pathologic neurologic traits of one of parental strains

Mice from the earlier developed recombinant inbred strains (RIS), which were derived by crossing 101/HY mice (carrying the mut-1 allele determining increased susceptibility to the mutagenic action of alkylating compounds) with C3H/Sn mice (lacking this trait), were tested for the presence of two neurological pathologies, audiogenic epilepsy and splitting of pyramidal cell layer of the CA3 hippocampal field (specific only to 101/HY mice). It was demonstrated that segregation of RIS relative to these traits was independent from the presence or absence of the mut-1 allele. These findings suggested the appearance of mut-1-independent mutations in the 101/HY mice, which resulted in the development of neurological pathologies. The appearance of such mutations can be the consequence of the genetic repair defects, earlier observed in the mice with this genotype.     

Imaging new neurons in vivo: a pioneering tool to study the cellular biology of depression?

Hippocampal neurogenesis has been implicated in the pathogenesis of and recovery from depression. However, most of the underlying studies were endpoint investigations in experimental animals yielding conflicting results, and it has been under debate to which extent these results could be transferred to human patients. Now, researchers have developed a powerful new tool to address these questions by a non-invasive method in humans and animals in vivo, using magnetic resonance spectroscopy to detect a biomarker for proliferating progenitor cells that give rise to new neurons. This makes it possible to study the role of neural progenitor cells in a wide variety of human brain disorders.     

Control of allergic rhinitis and asthma test – a formal approach to the development of a measuring tool

Background

The concurrent management of allergic rhinitis and asthma (ARA) has been recommended by Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines. However, a tool capable of assessing simultaneously the control of upper and lower airways diseases is lacking.

Aim

To describe the studies conducted to design the control of ARA test (CARAT) questionnaire.

Methods

We performed a literature review to generate a list of potentially important items for the assessment of control of ARA. A formal consensus development process, that used an innovative web-based application, was designed – 111 experts in ARA and 60 patients participated. At the final consensus meeting, 25 primary and secondary care physicians formulated the questions and response options. A qualitative feasibility study (n = 31 patients) was conducted to evaluate the comprehensibility of the questionnaire while testing two different designs.

Results

Thirty-four potentially important items were identified. All the steps of the consensus process were completed in 2.5 months. The opinions of experts and patients lead to the formulation of 17 questions. At the feasibility study the instructions and wording problems were corrected and a semi-tabular format was chosen.

Conclusion

A tool to measure the control of allergic rhinitis and asthma was developed using a comprehensive set of methodological steps ensuring the design quality and the face and content validity. Additional validation studies to assess the psychometric properties of the questionnaire have started.     

Identification of the development stage—specific factors in mouse fetal liver binding to the human β—globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development,the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared.By using DNase I footprinting and gel mobility shift assays,the binding of protein factors in these extracts to the human β-globin promoter was analyzed.The differences in the binding patterns of protein factors during development were observed.An erythroid-specific and stage-specific nuclear protein in the nuclear extrace from d 18 mouse fetal liver was identified,which can bind to the sequence(from-66bp to-90bp) of human β-globin promoter.We therefore speculate that the function of this cis-acting element may be similar to stage selector element(SSE) in chicken β^A-promoter.     

How does transferrin overcome the in vitro block to development of the mouse preimplantation embryo?

Transferrin promotes development of mouse embryos through the two-cell block in vitro. Uptake of transferrin into blastocysts was shown to occur by both receptor-mediated and nonspecific pathways, but neither pathway was used to a detectable extent by embryos before the eight-cell stage. Conversely, the dialysis of culture medium, non-permissive for development through the two-cell block, against a solution of transferrin rendered it capable of supporting development. It was therefore concluded that transferrin exerts its supportive effect on development in vitro via its chelating effects.     

Origin of genetic variation: regulation of genetic recombination in the higher organisms — a theory

Summary Recent studies in the fungi, particularly Neurospora and Schizophyllum, have revealed a number of genetic features which, viewed in conjunction with earlier observations on other organisms, form a pattern, or model, which appears to be basic to the control of recombination in all eukaryotes, including higher organisms. It is assumed that the control is exercised on mechanisms that produce new alleles through recombination, as understood in broad terms and including such a likely phenomenon as gene conversion, which may or may not involve crossing-over, as well as equal and unequal crossing-over. The recombination may thus occur between alleles in either the homozygous or heterozygous condition. In the model, regulatory genes and breeding behaviour are integrated into one self-regulatory system controlling the production of new genetic variation.The model is based on the following five general features, largely substantiated by the results in Neurospora and Schizophyllum: 1) The frequency of recombination in a particular chromosomal region is controlled by specific regulatory genes (rec). 2) There may be a number of such specific, regulatory genes responsible for recombination in a given region. 3) A rec. locus may influence recombination in more than one region. 4) The regulatory genes have no specific physical relationship with the region(s) they control, and are usually located at random in the genome. 5) Of the allelic forms of the regulatory genes it is always the dominant gene which suppresses recombination and the recessive gene which increases recombination. The rec system is epistatic to other genetic elements jointly involved in the overall control of recombination in a specific region. It is suggested that usually the control of recombination in a given region is exercised, cumulatively, by the balance of the dominant and recessive genes of the specific rec loci in the organism. Outbreeding, with the associated high heterozygosity of the regulatory rec loci, virtually switches off recombination, producing few new variations. Inbreeding produces homozygosity of these loci, resulting in certain individuals which will have a considerable number of their regulatory loci in the homozygous recessive condition and in which recombination will be switched on, producing new variation at a high frequency. Inbreeding is thus an integrated, evolutionary system of considerable importance, and is not a degenerate dead end, as many investigators have previously thought.The model has another compensatory function in evolution. In major loci, or in an operon, where there are structural genes and closely linked operator genes, as exemplified by the S locus, there are indications that the present model is concerned with the regulation of both structural and operator genes. The consequences of the model in the two classes of genes, however, are in direct contrast to each other: High heterozygosity which is instrumental in switching off recombination, and which is therefore helpful in maintaining stability in the structural gene, is conducive to functional variation of the operator gene; and high homozygosity, which is instrumental in switching on recombination, and which is therefore helpful in producing variation in the structural gene, is conducive to the stability of the operator gene.This model of the control of genetic variation in a specific chromosomal region is significant in development as well as in evolution, and throws light on a number of hitherto intractable problems peculiar to the higher organisms. For example, the model is helpful in explaining: 1) the origin of new self-incompatibility alleles in the flowering plants; 2) the impressive speciation in the waif flora (and fauna) of the oceanic islands; 3) the presence of high genetic variability in inbreeding species of plants; 4) environmentally-induced heritable variation in certain plants; and 5) the genetic mechanism of antibody diversity in animals.     

The shape of things to come in the study of the origin of species?

Perhaps Darwin would agree that speciation is no longer the mystery of mysteries that it used to be. It is now generally accepted that evolution by natural selection can contribute to ecological adaptation, resulting in the evolution of reproductive barriers and, hence, to the evolution of new species (Schluter & Conte 2009 ; Meyer 2011 ; Nosil 2012 ). From genes that encode silencing proteins that cause infertility in hybrid mice (Mihola et al. 2009 ), to segregation distorters linked to speciation in fruit flies (Phadnis & Orr 2009 ), or pollinator‐mediated selection on flower colour alleles driving reinforcement in Texan wildflowers (Hopkins & Rausher 2012 ), characterization of the genes that drive speciation is providing clues to the origin of species (Nosil & Schluter 2011 ). It is becoming apparent that, while recent work continues to overturn historical ideas about sympatric speciation (e.g. Barluenga et al. 2006 ), ecological circumstances strongly influence patterns of genomic divergence, and ultimately the establishment of reproductive isolation when gene flow is present (Elmer & Meyer 2011 ). Less clear, however, are the genetic mechanisms that cause speciation, particularly when ongoing gene flow is occurring. Now, in this issue, Franchini et al. ( 2014 ) employ a classic genetic mapping approach augmented with new genomic tools to elucidate the genomic architecture of ecologically divergent body shapes in a pair of sympatric crater lake cichlid fishes. From over 450 segregating SNPs in an F2 cross, 72 SNPs were linked to 11 QTL associated with external morphology measured by means of traditional and geometric morphometrics. Annotation of two highly supported QTL further pointed to genes that might contribute to ecological divergence in body shape in Midas cichlids, overall supporting the hypothesis that genomic regions of large phenotypic effect may be contributing to early‐stage divergence in Midas cichlids.     

In search of expression bottlenecks in recombinant CHO cell lines—a case study

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells’ quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell’s productivity.     

A genetic characterization of differences in the sensitivity to radiation-induced malformation frequencies in the mouse strains Heiligenberger,C57Bl,and Heiligenberger × C57Bl

We studied the frequency of malformations induced in two mouse strains (Heiligenberger, C57Bl/6J) by exposure to x-rays 3 h after conception. Whereas there was a high number of malformed fetuses in Heiligenberger mice (mostly gastroschises) on day 19 of pregnancy, C57Bl did not respond to radiation exposure shortly after conception with an increased frequency of malformed fetuses. Cross-breeding of both strains revealed that no statistically significant increase in radiation-induced malformations was obtained in the F₁ fetuses when the father was Heiligenberger and the mother C57Bl. In the opposite case (Heiligenberger mother, C57Bl father a small but statistically significant increase was observed.     

The genetic control of tolerance to aluminum toxicity in the ‘Essex’ by ‘Forrest’ recombinant inbred line population

Aluminum (Al) toxicity to plant roots is a major problem of acidic soils. The main chemical reaction involved is Al hydrolysis. Application of lime or nitrate fertilizers to raise soil pH reduces Al toxicity but not as economically as a plant genotypes with natural tolerance against this stress. Ammonium fertilization of crops and assimilation of ammonium (even that derived from dinitrogen) are particularly acidifying of the root zone. The aims of the present study were to find genotypes of soybean tolerant to aluminum stress and identify QTL underlying that trait. Used were recombinant inbred lines (RILs) derived from the cross of ‘Essex’ by ‘Forrest’. RILs were grown in a greenhouse for 3 weeks and then transferred to hydroponics in a growth chamber. Root lengths (RL) were measured before and 72 h after Al treatment. RL before and after Al treatment were measured and used to calculate root tolerance index (RTI) and relative mean growth (RMG). RILs 1, 85, 40 and 83 had significant (P < 0.005) tolerance to Al stress judged by RL after Al, RTI and RMG. Eleven minor but significant marker–trait associations (P < 0.05) were detected using one-way ANOVA but only two major loci were significant in composite interval maps (LOD >3.0). The QTL on linkage group F (chromosome 13) was in the interval Satt160–Satt252 with a peak at 24 cM (peak LOD was 3.3). The QTL underlay 31% of trait variation and the Essex allele provided an additional 1.61 cm of root growth over 72 h in the presence of Al. The QTL on linkage group C2 (probably chromosome 4) was in the interval from Satt202 to Satt371 with a peak at 3.2 cM (peak LOD was 14.7). The QTL underlay 34% of trait variation or 1.81 cm of growth over 72 h in the presence of Al. Both loci encompassed genes implicated in citrate metabolism, a method of aluminum detoxification known to vary among soybean cultivars. Two major loci and at least nine minor loci were inferred to underlie tolerance to Al. RILs and markers may be used to select alleles that increase tolerance to soybean against Al stress.     

Captive breeding—a useful tool in the preservation of biodiversity?

Within the next decades species extinction may eliminate between 20 and 50% of the Earth's species. Captive breeding has often been claimed to be a useful tool in preservation of biodiversity. The role of zoos in conservation work and the value of captive breeding are discussed; the latter exemplified by the Peregrine Falcon (Falco peregrinus) Programme and the Arabian oryx (Oryx leucoryx) Programme. Captive breeding programmes are very resource demanding and can only be afforded for a very small number of species, which limits their value significantly. Zoos deal mainly with vertebrates, but these comprise less than 3% of the described species, and although the 878 zoos considered hold more than 20 000 specimens of 140 threatened mammal species, they probably only contribute to the conservation of 20 full species. The situation for birds, reptiles and amphibians is even worse. Zoos face serious problems with minimum viable population sizes and hybridization. However, zoos can make a major contribution to preservation of biodiversity through educating and informing the public. Today, where the crisis of extinction of species has reached such daunting dimensions, captive breeding and otherex situ conservation tools should be the last resort for preserving biodiversity, and captive breeding must not become an excuse to avoid dealing with preservation of habitats.     

Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?

There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection.     

The Wageningen Rhizolab — a facility to study soil-root-shoot-atmosphere interactions in crops

A research facility is described for the integrated study of soil-root-shoot-atmosphere relationships in crops. The Wageningen Rhizolab has been in use since 1990, and consists of two rows, each with eight below-ground compartments aligned along a corridor. A rain shelter automatically covers the experimental area at the start of rainfall. Compartments are 125 cm × 125 cm and 200 cm deep. Each compartment has a separate drip irrigation system. Crop canopy photosynthesis, respiration, and transpiration can be measured simultaneously and continuously on four out of eight compartments at a time. Each compartment can be filled with a selected soil material (repacked soil) and is accessible from the corridor over its full depth. Multiple sensors for measuring soil moisture status, electrical conductivity, temperature, soil respiration, trace gases and oxygen are installed in spatial patterns in accordance with the requirements of the experiments. Sensors are connected to control and data-acquisition devices. Likewise, provisions have been made to sample manually the soil solution and soil atmosphere. Root observation tubes (minirhizotrons) are installed horizontally at depth intervals ranging from 5 cm (upper soil layers) to 25 cm (below 1 m). The facility is at present in use to study growth and development of vegetation (crops) in relation to drought, nutrient status, soil-borne diseases, and underground root competition. One important application is the study of elevated CO₂ concentration and climate change and the way they affect crops and their carbon economy. Growth and development of field grown vegetables and winter cover crops are also evaluated. The common aspect of those studies is to gain a better understanding of crop growth under varying environmental conditions, and to collect datasets that may help to improve mechanistic crop growth simulation models that can address suboptimal growth conditions.     

Prepulse inhibition （PPI） of tactile startle response in recombinant congenic strains of mice： QTL mapping and comparison with acoustic PPI

Prepulse inhibition (PPI) of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems.We analyzed the tactile startle response (TSR) and PPI of TSR (tPPD,using light as a prepulse stimulus,in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them.Parental strains were significantly different for TSR,but were comparable for tPPI.Among the congenic strains,variation for TSR was significant in both genetic backgrounds,but that of tPPI was significant only for the C57BL/6J background.Provisional mapping for loci modulating TSR and tPPI was carded out.Using mapping data from our previous study on acoustic startle responses (ASR) and PPI of ASR (aPPI),no common markers for aPPI and tPPI were identified.However,some markers were significantly associated with both ASR and TSIL at least in one genetic background.These results indicate cross-modal genetic regulation for the startle response but not for PPI,in these mouse strains.     

Histochemical study of the pre—and postnatal development of acetylcholinesterase in the rat spinal cord

The distribution of acetylcholinesterase(AChE)-positive structures in the developing rat spinal cord was studied with AChE-histochemistry.AChE-positive perikarya were first seen on embryonic day 14(E14) in the ventrolateral portion of the spinal cord.From that time onward.AChE=containing cells appeared gradually in the intermediate gray,dorsal horn and lateral spinal nucleus of the spinal cord in a ventral-to-dorsal,and lateral-to-medial order.No obvious rostral-to-caudal sequence was found.At birth,the distribution pattern of AChE-positive perikarya was basically similar to that in adults.After birth a dramatic increase in the AChE staining intensity extended from postnatal day 5(P5) to postnatal day 21(P21),In addition,two phases of transient AChE staining were observed in the external surface of the dorsal horn from embryonic day 15(E15) to embryonic day 21(E21) and in the marginal layer from embryonic day 21(E21) to postnatal day 14(P14),respectively.