A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail.

A quantitative trait loci (QTL) study was undertaken to identify genome regions involved in the control of fearfulness in Japanese quail (Coturnix japonica). An F2 cross was made between two quail lines divergently selected over 29 generations on duration of tonic immobility (DTI), a catatonic-like state of reduced responsiveness to a stressful stimulation. A total of 1065 animals were measured for the logarithm of DTI (LOGTI), the number of inductions (NI) necessary to induce the immobility reaction, open-field behaviour including locomotor activity (MOVE), latency before first movement (LAT), number of jumps (JUMP), dejections (DEJ) and shouts (SHOUT), corticosterone level after a contention stress (LOGCORT) and body weight at 2 weeks of age (BW2). A total of 310 animals were included in a genome scan using selective genotyping with 248 AFLP markers. A total of 21 suggestive or genome-wide significant QTL were observed. Two highly significant QTL were identified on linkage group 1 (GL1), one for LOGTI and one for NI. In the vicinity of the QTL for LOGTI, a nearly significant QTL for SHOUT and a suggestive QTL for LAT were also identified. On GL3, genome-wide significant QTL were observed for JUMP and DEJ as well as suggestive QTL for LOGTI, MOVE, SHOUT and LAT. A significant QTL for BW2 was observed on GL2 and a nearly significant one on GL1. These results may be useful in the understanding of fearfulness in quail and related species provided that fearfulness has the same genetic basis.

Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue.

Lymphoid follicles cluster in the terminal rectum of various animal species and of man and hence this site may be important in the development of immune responses to pathogens. For the induction of immune responses at mucosal sites, interplay is required between various cell types performing functions ranging from antigen-sampling cells via antigen-presenting cells to antigen-specific lymphocytes. Therefore, we have characterised the cell populations and relevant functioning of follicle-associated epithelium (FAE) and associated follicles in the terminal portion of rectum in cattle as a representative mammal. Immunohistochemical studies of this region identified immune cell subsets (CD4+, CD8+, WC 1+gammadelta, CD2+, CD 21+ and CD 40+ cells) characteristic of an immune-inductive site. Examination of FAE identified a subset of cells with structural and functional features of antigen-sampling M-cells. Cells of the FAE and adjacent follicle-associated crypts expressed vimentin and a subset of these cells internalised microparticles, a further attribute of M-cells. The FAE cells were phenotypically heterogeneous and therefore the function and phenotype of these cell subsets requires further characterisation, particularly with respect to their potentially important role in the interaction of hosts with pathogens and the development of immune responses.

Fearfulness and performance related traits in selected lines of Japanese quail (Coturnix japonica).

Fearfulness and economic traits were studied in three lines of Japanese quail. Two of the lines were of the same genetic origin and were subjected to divergent selection for the duration of tonic immobility (TI), a measure of fearfulness. Birds were selected for long (LTI) or short (STI) duration of TI. The third line (DD) was of a different genetic origin and had been selected for early egg production. Fear, growth, residual feed intake, and measures of egg composition and production varied among lines. The distribution of TI in Line DD was closer to that from Line STI. Residual feed intake and shell content were lowest in the DD line. The DD birds laid more broken eggs than quail of the other lines. The STI line birds had higher BW and laid more, but smaller, eggs than LTI line birds. Eggs laid by LTI line birds had higher albumen content, but lower percentage shell, than those laid by STI line birds. When all traits were considered together, there was an overall tendency for STI line birds to out perform LTI birds with DD line birds showing intermediate performance. This finding supports the notion that there is a relationship between fearfulness and productivity. However, the skewed distribution of TI precluded estimation of correlation with production traits in the LTI and STI lines. No significant relationships among fear and production-related traits were found in the DD line, which contradicts the notion that fearfulness and production are related.

The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells.

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescent murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate well. Permeabilization of the nuclear envelope rescues the ability of quiescent nuclei to replicate in the extract. We show that origin replication complex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all present in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex from Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mouse homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiescent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins from quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cells. Surprisingly, intact quiescent nuclei fail to bind exogenously added XCdc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even though G1- or S-phase nuclei still replicate in these extracts. Our results identify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to replicate in vivo and in Xenopus egg extracts.

Effect of group disruption on social behaviour in lines of Japanese quail (Coturnixjaponica) selected for high or low levels of social reinstatement behaviour.

Japanese quail of lines selected for high (HSR) or low (LSR) levels of social reinstatement (SR) behaviour were reared in groups of three. At 3 and 6 weeks of age, one group member was replaced with an unfamiliar conspecific bird. Behaviour following introduction of the intruder was similar at both ages. Quail of both lines spent more time immobile after introduction of the intruder than before its introduction. In the LSR, but not the HSR line, inter-individual distances between intruders and other chicks, were greater than those between familiar chicks. Birds of both lines pecked more between unfamiliar than between familiar birds. Pecks, not associated with escape or avoidance by the pecked bird, were more frequent in HSR groups. Selection for HSR behaviour appears to have increased non-aggressive exploration of intruder whereas selection for LSR behaviour has decreased interest in or increased shunning of intruders.

Minichromosome maintenance proteins as biological markers of dysplasia and malignancy.

Dysplasia, an intermediate stage in the progression from normal tissue to neoplasia, is defined morphologically by a loss of normal orientation between epithelial cells, with changes in cellular and nuclear shape and size. However, little is known about the functional properties of dysplastic cells, including their replicative state, largely due to a lack of available biological markers. We have used novel antibodies against minichromosome maintenance (MCM) proteins to examine the proliferative status of a range of histological lesions and to characterize dysplastic cells in functional terms. Immunoperoxidase staining was used to localize the MCM proteins, components of the prereplicative complex that is essential for initiating eukaryotic DNA replication. These proteins are down-regulated in cells undergoing differentiation or quiescence and, thus, serve as specific markers for proliferating cells. In normal and some reactive tissues, MCM expression was present only in restricted proliferative compartments, consistent with our published findings in the uterine cervix. In dysplastic and malignant tissues, in contrast, MCM proteins were expressed in the majority of cells, extending to surface layers of dysplastic stratified epithelia. In carcinomas, the frequency of expression of MCM proteins showed an inverse correlation with the degree of tumor differentiation. Thus, we suggest that dysplastic cells may be characterized in functional terms as remaining in cell cycle, due to deregulation of normal controls over cell proliferation. Antibodies against MCM proteins have potential clinical applications, for example, in the assessment of tumor prognosis in histological sections and the identification of proliferating cells in clinical samples using biochemical or cytological assays.

Inter-individual distances during open-field tests in Japanese quail (Coturnix japonica) selected for high or low levels of social reinstatement behaviour.

Inter-individual distances (IIDs), during open-field tests, were measured in single sex pairs of quail of lines divergently selected for high (HSR) or low (LSR) levels of social reinstatement (SR) behaviour. Tests were carried out when the chicks were 1-, 3- and 6-weeks of age. IIDs were established within 1 min and remained stable thereafter. In HSR and mixed line pairs, but not LSR line pairs, IIDs increased with age. IIDs were shorter in HSR line pairs than in LSR or mixed line pairs at 1- and 3-weeks of age but not at 6-weeks of age when IIDs were similar in all pair-types. At 1- and 3-weeks of age, IIDs in mixed line pairs were intermediate to those in HSR and LSR line pairs. The sex of the pair-members did not influence IIDs. Previous studies have shown that selection for SR behaviour, in quail chicks, has effects that persist into later life and influence characters not present in the birds' behavioural repertoire at the time of testing for the purposes of selection. Such behaviour patterns include aggression, sexual behaviour and socio-sexual proximity behaviour. All of these behaviour patterns are expressed at higher levels in HSR line birds than LSR line birds. Differences in IIDs between HSR and LSR line birds may, therefore, be due to an interaction between environmental and genetic effects. In young HSR chicks, social reinstatement is the primary motivation and IIDs are short. However, as the HSR line chicks' age, IIDs reflect an interaction between social attraction and aggression related avoidance behaviour that tends to increase IIDs. In LSR line chicks aggression and social motivation are low at all ages and IIDs tend to remain stable.

Improved cervical smear assessment using antibodies against proteins that regulate DNA replication.

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

Cdc6 protein causes premature entry into S phase in a mammalian cell-free system.

We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.

Meat quality traits and muscle characteristics in high or low fear lines of Japanese quails (Coturnix japonica) subjected to acute stress.

1. The influence of acute stress before slaughter on muscle and meat quality was studied in adult male quail from lines divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI) and from the unselected (C) control line. 2. When subjected to acute stress, birds from the long TI line responded differently to those of the control or the low fear lines. LTI birds exhibited: a higher plasma creatine kinase and a smaller increase in plasma corticosterone levels, a higher pHu and drip loss values for breast meat associated with no differences in L*a*b* colour values; a higher percentage of fast glycolytic myofibres (IIb) and correspondingly (correlate) higher glycolytic (activity) in the pectoralis major (PM) muscle, and consequently lower oxidative enzyme activity in the PM muscle. 3. It is concluded that acute stress affected muscle metabolism differently in the STI, C and LTI lines. Hence, selection for TI, which is correlated with underlying fearfulness, can indirectly lead to differences in meat quality.

Protein kinase inhibition in G2 causes mammalian Mcm proteins to reassociate with chromatin and restores ability to replicate.

Intact nuclei from G2-phase mammalian cells will replicate their DNA in Xenopus egg extract if they are preexposed to the protein kinase inhibitor 6-dimethylaminopurine in vivo (Coverley et al., Exp. Cell Res. 225, 294-300, 1996). Here, we demonstrate that this competence to rereplicate is accompanied by alterations in the subcellular distribution of the Mcm family of proteins, which are implicated in replication licensing (Hennessy et al., Genes Dev. 4, 2252-2263, 1990; Kubota et al., Cell 81, 601-609, 1995; and Chong et al., Nature 375, 418-421, 1995). All family members reassociate with chromatin in G2 cells and this correlates closely with regeneration of replication competence. Moreover, newly bound Mcm proteins are functional for replication because, unlike untreated G2 nuclei, replication of treated G2 nuclei in vitro occurs independent of the Xenopus Mcm protein complex. These observations show that the postreplicative state is actively maintained in G2 cells by a protein kinase(s) which regulates the behavior of Mcm family proteins.

Place preferences of Japanese quail given a permanent choice between a social or a non-social but enriched situation.

Japanese quail from two lines selected for high (HSR) or low (LSR) levels of social reinstatement behaviour were permanently placed from 2 days to 6 weeks of age in a situation where one tested animal had the choice between being in social contact with a stimulus animal in a bare compartment of the cage or out of visual contact with the stimulus animal in a relatively rich environment containing food, water and wood shavings. The young (1 or 2 weeks of age) quails spent most of their time (over 80%) in the social compartment, about 10% feeding and drinking, and about 10% performing other activities in the non-social compartment. Hardly any differences appeared between the two lines when 1 or 2 weeks old, but at 4 weeks of age the LSR quails started to decrease the time spent in the social compartment and this phenomenon only appeared at 6 weeks of age and to a lesser extent in the HSR quails. At 4 and 6 weeks the differences between lines were significant. The lack of difference between the two lines in young quails can be explained by the very high social motivation expressed even by LSR quails in the conditions of the study. When they are older line differences are expressed. This change coincides with the time when the natural coveys start to disperse and also with the beginning of sexual development. These results confirm the hypothesis of François et al. (François, N., Mills, A.D., Faure, J.M., 1997. Inter-individual distances in Japanese quail (Coturnix japonica) selected for high or low levels of sociability. Behav. Process. (in press)) that line differences in social motivation persist into adult or near adult life and can still be demonstrated, provided the circumstances are such that aggression does not prevent close social contact.

The behavior of the Japanese or domestic quail Coturnix japonica.

This paper reviews the literature pertaining to the behavior of the Japanese or domestic quail Coturnix japonica. Details are given of the classification, characteristics, domestication and the economic and research potential of the species. Further sections deal with sensation and perception (including taste and smell, vision and hearing), maintenance behavior (including feeding and drinking, dust bathing and thermoregulation), development and aging (including vocalization, filial imprinting, sexual imprinting, fear and avoidance responses, sexual maturation and aging), adult learning (including habituation, instrumental conditioning, Pavlovian conditioning and observational learning), photoperiodism, reproductive behavior (including courtship and mating, hormonal control and ontogeny of sexual differentiation, and male and female sexual behavior), parental behavior (including nest-site selection and nest building, incubation behavior and its hormonal control, and hen-chick relationships), and aggressive behavior and dominance (including agonistic behavior and the hormonal control of aggressive behavior).

Differences in benzodiazepine binding in quail selectively bred for differences in tonic immobility.

We have previously found that quail selectively bred to exhibit long (LTI) or short (STI) tonic immobility responses to manual restraint differed with respect to the affinity of binding at the diazepam-sensitive benzodiazepine binding site. In the current study, binding at other components of the GABAA-benzodiazepine receptor complex was investigated. Whereas the lines did not differ in either number or affinity of GABAA receptors, we found that GABA caused significantly greater enhancement of [3H]flunitrazepam binding in the forebrains of LTI than in STI birds. There was also significantly higher binding to the diazepam-insensitive component (alcohol binding site) of the GABAA-benzodiazepine complex in the forebrain of the LTI line. It is not known, however, whether this difference in the lines is due to differences in number or affinity of these sites. It is discussed whether these manifold differences between the lines in binding at the forebrain sites of the GABAA-benzodiazepine complex could be founded on subunit differences, and whether differences in benzodiazepine binding could underlie the genetically determined behavioural differences in tonic immobility.

Hyperphosphorylation of nucleoplasmin facilitates Xenopus sperm decondensation at fertilization.

Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus.

Social discrimination in Japanese quail Coturnix japonica chicks genetically selected for low or high social reinstatement motivation.

Japanese quail chicks of two lines genetically selected for low levels of social reinstatement behaviour (LSR) or high levels of social reinstatement behaviour (HSR) were housed in groups of three. The approach/avoidance tendencies of an individual chick from each group towards a familiar cagemate and a stranger from another group placed at opposite ends of a runway were measured at 7 days of age. Quail chicks of both genetic lines preferentially approached, spent longer near, and showed shorter mean distances from the cagemate than the stranger. Irrespective of stimulus type, the HSR quail generally showed shorter approach latencies, spent longer within 15 cm of the goal boxes, made more entries into these areas, and travelled further during the test than did their LSR counterparts. These findings demonstrated that Japanese quail chicks, of two genetic lines reared in small groups, successfully discriminated between familiar cagemates and strangers. They also suggest that there are no straightforward relationships between underlying social motivation and social preferences.

The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts.

BACKGROUND: A complex of MCM proteins is implicated in ensuring that DNA replicates only once in each cell cycle, by 'replication licensing'. The nuclear membrane is also implicated in replication licensing, but the relationship between the MCM proteins and the nuclear membrane is unclear. Here, we investigate the relationship between XMCM3 (a component of the Xenopus MCM complex), nuclear envelope permeability and the initiation of DNA replication once per cell cycle. RESULTS: Our results show that the nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but that it does prevent the binding of XMCM3 to chromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication. XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' that is excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane. CONCLUSIONS: The Xenopus MCM complex contains homologues of yeast MCM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results we present a two-stage model for replication licensing.

Distinct functions for the two importin subunits in nuclear protein import.

The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran. Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref. 16). As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus. In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.