Dairy goats adjust their meal patterns to the fibre content of the diet.

Few studies have investigated how meal patterns of ruminants are affected by diet fibre content. Dairy goats (N = 32) in late lactation and early gestation were housed in eight groups of four goats, with all combinations of breed (Alpine and Saanen) and lactation number (1 and 2) represented in each group. Each goat had access to its own individual feed trough placed on a weigh scale with data logged automatically. All goats were fed the same total mixed ration (TMR; 30% concentrate and 44.6% NDF in DM) ad libitum for a control period of 22 days. Using the same feed ingredients, half of the groups were then offered a High fibre diet (20% concentrate; 47.3% NDF), and the other half a Low fibre diet (40% concentrate; 41.5% NDF) for a treatment period of 16 days. Daily meal patterns (meal frequency, duration and size, feeding rate, daily feed intake and daily feeding time) were computed for each animal using a meal criterion of 8 min. The last 10 days for each period (control and treatment) were used to calculate individual period means and individual differences between the two periods. During the control period, the goats ate on average 12.1 ± 0.49 meals/day, consuming 4.2 ± 0.10 kg fresh TMR daily. When the ration changed, all measures of feeding behaviour except meal size changed asymmetrically for the goats on the two diets. Goats fed the High fibre diet reduced their meal frequency by 10%, and the first meal after feed distribution lasted 11% longer, leading to a 9% reduction in feeding rate and no significant changes in daily feed intake and daily feeding time. Goats on the Low fibre diet did not significantly change their meal frequency or meal size, but the combined changes nevertheless led to a 9% increase in daily feed intake. On the Low fibre diet, goats were able to increase their feeding rate by a third, leading to a reduction in meal durations, thus reducing daily feeding time by 13%. Goats adapt their feeding behaviour to the fibre proportion of the offered diet, with more changes when fibre content is lowered, which needs to be taken into account when comparing phenotypes and adaptability of small ruminants to different diets.

Short communication: Calving site selection of multiparous, group-housed dairy cows is influenced by site of a previous calving.

A calving cow and her newborn calf appear to have an attracting effect on periparturient cows, which may potentially influence the functionality of future motivation-based calving pen designs. In this pilot study we examined whether calving site selection of group-housed Holstein dairy cows was affected by the site of a previous calving. Ten multiparous cows moved to 1 of 2 group pens 11 (range = 4-27) d before calving were included. Each pen consisted of an open area (9 × 9 m) connected to 6 secluded areas (4.5 × 3 m each), where cows could move freely between all areas. Time of calving, location of the breaking of the amniotic sac, as well as the place of birth were recorded. In all but 1 case cows calved within a distance of 1 cow length from where the previous calving took place, suggesting that the cows did not select calving site at random. These preliminary observations indicate that choice of calving site may be affected by the site of a previous calving, potentially explained by the presence of amniotic fluids.

Review: Drinking water for liquid-fed pigs.

Liquid feeding has the potential to provide pigs with sufficient water to remain hydrated and prevent prolonged thirst. However, lack of permanent access to fresh water prevents animals from drinking when they are thirsty. Moreover, individual differences between pigs in a pen may result in uneven distribution of the water provided by the liquid feed, leading to some pigs being unable to meet their water requirements. In this review, we look at the need for and provision of water for liquid-fed pigs in terms of their production performance, behaviour, health and welfare. We highlight factors which may lead to water ingestion above or below requirements. Increases in the need for water may be caused by numerous factors such as morbidity, ambient temperature or competition within the social group, emphasising the necessity of permanent access to water as also prescribed in EU legislation. The drinkers can be the target of redirected behaviour in response to feed restriction or in the absence of rooting materials, thereby generating water losses. The method of water provision and drinker design is critical to ensure easy access to water regardless of the pig's physiological state, and to limit the amount of water used, which does not benefit the pig.

Welfare of organic laying hens kept at different indoor stocking densities in a multi-tier aviary system. I: egg laying, and use of veranda and outdoor area.

Multi-tier aviary systems are becoming more common in organic egg production. The area on the tiers can be included in the net area available to the hens (also referred to as usable area) when calculating maximum indoor stocking densities in organic systems within the EU. In this article, results on egg production, laying behaviour and use of veranda and outdoor area are reported for organic laying hens housed in a multi-tier system with permanent access to a veranda and kept at stocking densities (D) of 6, 9 and 12 hens/m2 available floor area, with concomitant increases in the number of hens per trough, drinker, perch and nest space. In a fourth treatment, access to the top tier was blocked reducing vertical, trough and perch access at the lowest stocking density (treatment D6x). In all other aspects than stocking density, the experiment followed the EU regulations on the keeping of organic laying hens. Laying percentage was significantly lower (P<0.05) in D12 compared with the other stocking densities (90.6% v. 94.3% (± 0.7)), most likely due to the concomitant reduction in nest space and drinker availability per hen. No systematic effects of density were found on other laying variables (egg weight, eggs laid outside nests, aviary side preferences). Number of hens using the veranda increased with stocking density. Hens primarily used the range near the house (within 50 m) and hens kept at the lowest stocking density and the smallest group size appeared to use the outdoor area more extensively, based on an assessment of vegetation cover (P<0.05). For the measures reported here, the welfare consequences of increased stocking density were assessed to be minor; additional results are reported in the associated article (Steenfeldt and Nielsen, 2015).

Welfare of organic laying hens kept at different indoor stocking densities in a multi-tier aviary system. II: live weight, health measures and perching.

Multi-tier aviary systems, where conveyor belts below the tiers remove the manure at regular intervals, are becoming more common in organic egg production. The area on the tiers can be included in the net area available to the hens (also referred to as usable area) when calculating maximum indoor stocking densities in organic systems within the EU. In this article, results on live weight, health measures and perching are reported for organic laying hens housed in a multi-tier system with permanent access to a veranda and kept at stocking densities (D) of 6, 9 and 12 hens/m2 available floor area, with concomitant increases in the number of hens per trough, drinker, perch and nest space. In a fourth treatment, access to the top tier was blocked reducing vertical, trough, and perch access at the lowest stocking density (D6x). In all other aspects than stocking density, the experiment followed the EU regulations on the keeping of organic laying hens. Hen live weight, mortality and foot health were not affected by the stocking densities used in the present study. Other variables (plumage condition, presence of breast redness and blisters, pecked tail feathers, and perch use) were indirectly affected by the increase in stocking density through the simultaneous reduction in access to other resources, mainly perches and troughs. The welfare of the hens was mostly affected by these associated constraints, despite all of them being within the allowed minimum requirements for organic production in the EU. Although the welfare consequences reported here were assessed to be moderate to minor, it is important to take into account concurrent constraints on access to other resources when higher stocking densities are used in organic production.

Effects of ambient temperature and early open-field response on the behaviour, feed intake and growth of fast- and slow-growing broiler strains.

Increased activity improves broiler leg health, but also increases the heat production of the bird. This experiment investigated the effects of early open-field activity and ambient temperature on the growth and feed intake of two strains of broiler chickens. On the basis of the level of activity in an open-field test on day 3 after hatching, fast-growing Ross 208 and slow-growing i657 chickens were allocated on day 13 to one of the 48 groups. Each group included either six active or six passive birds from each strain and the groups were housed in floor-pens littered with wood chips and fitted with two heat lamps. Each group was fed ad libitum and subjected to one of the three temperature treatments: two (HH; 26°C), one (HC; 16°C to 26°C) or no (CC; 16°C) heat lamps turned on. Production and behavioural data were collected every 2 weeks until day 57. For both strains, early open-field activity had no significant effects on their subsequent behaviour or on any of the production parameters measured, and overall, the slow-growing strain was more active than the fast-growing strain. Ambient temperature had significant effects on production measures for i657 broilers, with CC chickens eating and weighing more, and with a less efficient feed conversion than HH chickens, with HC birds intermediate. A similar effect was found for Ross 208 only for feed intake from 27 to 41 days of age. Ross 208 chickens distributed themselves in the pen with a preference for cooler areas in the hottest ambient temperature treatments. In contrast, the behaviour of the slow-growing strain appeared to be relatively unaffected by the ambient temperature. In conclusion, fast-growing broilers use behavioural changes when trying to adapt to warm environments, whereas slow-growing broilers use metabolic changes to adapt to cooler ambient temperatures.

Splitting the scotoperiod: effects on feeding behaviour, intestinal fill and digestive transit time in broiler chickens.

1. The aim of this study was to evaluate how splitting the dark period (scotoperiod) affects feeding behaviour and associated intestinal measures in broilers. 2. Ross 308 broilers were reared to 37 d in groups given either a daily 8-h continuous scotoperiod (DARK 8) or an intermittent light schedule with two equally spaced 4-h scotoperiods (DARK 4 + 4), which yielded the same total duration of darkness per 24 h. 3. Feeding behaviour was recorded weekly from 24-h video recordings of 24 groups each of 64 birds. Empty intestinal weights as well as their contents were measured weekly at 4 time points (n = 192). Digestive transit time was estimated on d 29 using a chromic oxide marker; production variables and the extent of foot pad dermatitis were also recorded. 4. In the 3 h prior to a scotoperiod, feeding activity increased in chickens from DARK 8 but not DARK 4 + 4. This increase was reflected in a higher relative content of the crop in DARK 8 at this time. 5. Immediately following the scotoperiod, feeding activity peaked and, although the chickens in DARK 4 + 4 expressed more feeding behaviour in the first 20 min after the scotoperiod, the chickens in DARK 8 had overall higher feeding activity across the day. However, DARK 4 + 4 had a higher feed intake and weight gain. The occurrence and severity of foot pad dermatitis was similar between treatments. 6. In conclusion, broilers modify their feeding behaviour according to the prevailing light schedule. Eight consecutive hours of darkness reduced growth, but did not affect overall feed conversion efficiency, and did not appear to exacerbate hunger or foot pad dermatitis to any great extent.

Proportion of insoluble fibre in the diet affects behaviour and hunger in broiler breeders growing at similar rates.

With a view to alleviate the feeling of hunger in broiler breeders, different types of fibre sources were used in high-fibre diets to increase feed quantity while limiting growth to industry recommended levels. Using scatter feeding, three diets (C1: commercial control diet, 1 × fibre content, 80% insoluble fibre (ISF); H2: 2 × fibre content, 89% ISF; and L2: 2 × fibre content, 71% ISF) were each fed to 10 groups of 16 broiler breeder chickens. Similar growth rates were obtained on different quantities of food with all birds reaching commercial target weight at 15 weeks of age. In a hunger test, birds fed C1 ate significantly faster and showed a higher compensatory feed intake than birds on diets H2 and L2, indicating that the two high-fibre diets did reduce the level of hunger experienced by the birds. Behavioural observations carried out at 14 weeks of age showed high levels of tail pecking in birds fed C1 and almost none in birds fed L2, whereas birds fed H2 were intermediate. Stereotypic pecking at fixtures was seen twice as frequently in birds fed C1. Birds on diet L2 displayed behavioural signs indicative of discomfort, and the high water usage on this diet created problems with litter quality. Birds on diet H2 continued to show foraging behaviour throughout the day, and were more frequently engaged in dust bathing and other comfort behaviour. This experiment indicates that high-fibre diets can alleviate the feeling of hunger currently experienced by broiler breeders, and a high ratio of ISF may improve the well-being of the birds.

Road transport of farm animals: effects of journey duration on animal welfare.

Transport of farm animals gives rise to concern about their welfare. Specific attention has been given to the duration of animal transport, and maximum journey durations are used in legislation that seek to minimise any negative impact of transport on animal welfare. This paper reviews the relatively few scientific investigations into effects of transport duration on animal welfare in cattle, sheep, horses, pigs and poultry. From the available literature, we attempt to distinguish between aspects, which will impair welfare on journeys of any duration, such as those associated with loading, and those aspects that may be exacerbated by journey time. We identify four aspects of animal transport, which have increasing impact on welfare as transport duration increases. These relate to (i) the physiological and clinical state of the animal before transport; and - during transport - to (ii) feeding and watering; (iii) rest and (iv) thermal environment. It is thus not journey duration per se but these associated negative aspects that are the cause of compromised welfare. We suggest that with a few exceptions, transport of long duration is possible in terms of animal welfare provided that these four issues can be dealt with for the species and the age group of the animals that are transported.

Feeding activity in groups of newly hatched broiler chicks: effects of strain and hatching time.

The feeding activity of 2 strains of broiler chickens was investigated during their first week of life in relation to their hatching time. Fast (Ross 308) and slow-growing (LB) strains were allocated to 1 of 3 (early, middle, or late hatch) single-strain groups of 80 to 100 as-hatched birds in 4 replicates divided into 2 time-separated blocks. Behavioral observations differed between blocks and were carried out at intervals on d 1 to 6, and the percentage of birds feeding (from trough or paper), drinking, or being otherwise active (block 2 only) were registered. A higher mortality caused by flip-over was seen among the late-hatching birds from the slow-growing strain. The percentage of birds engaged in feeding activity was similar for the 2 strains, but LB birds began to eat from the paper later and were observed eating from the trough less than Ross 308 birds, which in turn were less active than LB, especially in the early and middle hatch groups. Early hatch groups were observed feeding from the paper more than the middle and late hatch groups. Drinking behavior mirrored feeding from the trough, indicating that drinking was prandial. Within strain, no effect of hatch time was found on live weight at hatch, but the feeding behavior of early hatched birds led to a small, transient weight advantage on d 3 after hatch. The transition from feeding on paper to feeding only from the trough may have less effect on birds that feed from the trough sooner, such as the fast-growing strain.

Ultradian activity rhythms in large groups of newly hatched chicks (Gallus gallus domesticus).

A clutch of young chicks housed with a mother hen exhibit ultradian (within day) rhythms of activity corresponding to the brooding cycle of the hen. In the present study clear evidence was found of ultradian activity rhythms in newly hatched domestic chicks housed in groups larger than natural clutch size without a mother hen or any other obvious external time-keeper. No consistent synchrony was found between groups housed in different pens within the same room. The ultradian rhythms disappeared with time and little evidence of group rhythmicity remained by the third night. This disappearance over time suggests that the presence of a mother hen may be pivotal for the long-term maintenance of these rhythms. The ultradian rhythm of the chicks may also play an important role in the initiation of brooding cycles during the behavioural transition of the mother hen from incubation to brooding. Computer simulations of individual activity rhythms were found to reproduce the observations made on a group basis. This was achievable even when individual chick rhythms were modelled as independent of each other, thus no assumptions of social facilitation are necessary to obtain ultradian activity rhythms on a group level.

Effects of a calm companion on fear reactions in naive test horses.

REASON FOR PERFORMING STUDY: In fear-eliciting situations, horses tend to show flight reactions that can be dangerous for both horse and man. Finding appropriate methods for reducing fearfulness in horses has important practical implications. OBJECTIVES: To investigate whether the presence of a calm companion horse influences fear reactions in naive subject horses. HYPOTHESES: The presence of a habituated (calm) companion horse in a fear-eliciting situation can reduce fear reactions in naive subject horses, compared to subject horses with a nonhabituated companion (control). METHODS: Minimally handled (n = 36), 2-year-old stallions were used, 18 as subjects and 18 as companions. Companion horses (n = 9) were habituated to an otherwise frightening, standardised test stimulus (calm companions), whereas the rest (n = 9) of the companion horses remained nonhabituated (control companions). During the test, unique pairs of companion and subject horses were exposed to the test stimulus while heart rate and behavioural responses were registered. Subsequently, subject horses were exposed to the stimulus on their own (post test). RESULTS: Subject horses, paired with a calm companion horse, showed less fear-related behaviour and lower heart rate responses compared to subject horses with control companions. Results from the post test suggest that the difference between treatment groups remained in the subsequent absence of companion horses. CONCLUSIONS AND POTENTIAL RELEVANCE: It appears possible to reduce fear reactions in young, naive horses by allowing them to interact with a calm companion horse in fear-eliciting situations.

Foot pad dermatitis and hock burn in broiler chickens and degree of inheritance.

A total of 2,118 birds from 2 strains were allocated to 12 groups of 93 to 100 each in 2 time-separated replicates. The development of foot pad dermatitis (FPD) and hock burn (HB) were recorded weekly from d 8 to slaughter on a set sample of live animals (7 per group). In addition, feet and hocks of all birds were investigated at slaughter at either 4, 6 (fast-growing strain), 8, or 10 (slow-growing strain) wk of age. Lesions were scored for both the left and right foot and classified according to a scale from 1 (no lesion) to 9 (very severe lesions) for FPD and from 1 (no lesion) to 3 (very severe lesions) for HB. No FPD lesions and very few low-grade HB lesions were found in chickens from the slow-growing strain. In the fast-growing strain, the first signs of FPD and HB were seen in wk 2. The incidence of both types of lesions increased thereafter. Foot pad dermatitis was more frequent in females (49 vs. 36%, P < 0.05). Body weight did not affect FPD, but more HB were found at higher BW (P < 0.01). Egg weight influenced neither FPD nor HB. Variance and covariance components were analyzed using a multivariate animal model, in which scores for FPD and HB were transformed into logarithmic scale. The analyses were carried out using restricted maximum likelihood algorithm. Heritabilities were estimated to be 0.31 +/- 0.12 (SE) for FPD, 0.08 +/- 0.08 for HB, and 0.38 +/- 0.13 for BW. Genetic correlations among these traits were low and nonsignificant. Phenotypic correlation between BW and FPD was low and nonsignificant and between BW and HB was 0.17 +/- 0.05 (P < 0.01). The relative high heritability of FPD and the low genetic correlation to BW suggested that genetic selection against susceptibility to FPD should be possible without negative effects on BW gain.

Why women want prenatal ultrasound in normal pregnancy.

OBJECTIVES: To investigate women's reasons for requesting prenatal ultrasound in the absence of clinical indications. METHODS: A postal questionnaire was completed by 370 pregnant women with no apparent obstetric risk factors, who had expressed a desire to have ultrasound scanning in their current pregnancy. The women were asked to indicate, from a list of 12 items, their three most important reasons for wanting scanning. Ninety per cent of the women were in the first trimester of pregnancy, and 10% in the second trimester. RESULTS: The items most frequently identified as important reasons for ultrasound were to check for fetal abnormalities (60% of women), to see that all was normal (55%) and for own reassurance (44%). Lower income was related to wanting to see the baby (P = 0.028) and wanting an ultrasound picture (P = 0.017); higher income was related to checking that all was normal (P = 0.003) and for own reassurance (P = 0.015). Women in their first pregnancy were more likely to want themselves and the father to see the baby (P = 0.001); women who had given birth previously were more likely to want reassurance (P = 0.002), as were women with a previous miscarriage or induced abortion. Women who believed that the presence of fetal trisomy justifies abortion or who would vote for free abortion were more likely to want to know about abnormalities (P < 0.001 and P < 0.004, respectively). Women in the second trimester were more likely to want to check for abnormalities (P = 0.041) and appropriate fetal growth (P = 0.047) than those in the first trimester. CONCLUSIONS: It would appear that women in normal pregnancy have specific reasons for wanting prenatal ultrasound that are influenced by sociodemographic, obstetric and attitudinal factors.

Breast blisters in groups of slow-growing broilers in relation to strain and the availability and use of perches.

1. The relationship between perching behaviour, availability of perches and the incidence and severity of breast blisters in broilers was investigated together with a comparison between two slow-growing broiler strains. 2. Sixteen single strain groups (n = 60) of Labresse and i657 broilers were subjected throughout the experiment to one of three perch availabilities: 15 cm per bird (Labresse and i657), 7.5 cm per bird (i657 only), and 0 cm per bird (i657 only) with 4 replicates per treatment. The birds were housed indoors from one day old, and at 43 d of age 52 birds from each group were moved to outdoor housing facilities with access to grass-covered outdoor areas until slaughter at 84 d of age. 3. The use of perches was monitored via video recordings throughout the experimental period. The severity of breast blisters was recorded on a scale from 0 to 2 at slaughter. 4. Groups of i657 with 15 cm perch per bird used these more than groups with 7.5 cm perch per bird (19% vs 8% of birds perching at midnight). A positive association between access to perches and severe breast blisters (score 2) was found in the groups of i657, with odds ratios of 3.1 and 3.4 for 7.5 and 15.0 cm per bird, respectively, relative to the no-perch treatment. 5. Labresse were more likely to develop breast blisters than i657 (odds ratio 3.5), but used the perches less (0.1 vs 9.5 birds perching at midnight) and weighed less (2011 g vs 2246 g) than i657. 6. Males had a higher incidence of breast blisters than females (odds ratio 12.2), and this was most prominent in the Labresse strain (odds ratio 40.0). 7. In some broiler strains access to perches may be associated with an increase in the occurrence of severe breast blisters, but strain and sex of broiler chickens appear to have a much larger influence than access to perches on the incidence of breast blisters.

Feed and strain effects on the use of outdoor areas by broilers.

1. The use of outdoor areas by two broiler strains, known to have different growth rates and with access to either moderate (M) or low (L) energy feeds, was examined to assess the consequences for production, meat quality, health and welfare. 2. Sixteen groups of either Ross 208 (ROSS) or a Labresse cross (LAB) were fed either feed M or L. They were raised indoors until 42 d of age when groups of 102 birds were moved to outdoor houses with free access to an outdoor area. 3. The number of chickens standing and lying, respectively, in each of 5 zones in the outdoor area were recorded at intervals throughout the outdoor growth period until slaughter at 84 d of age. Production variables were measured, and gait, feather condition, litter quality, and dermal lesions on foot-pads and hocks were scored together with objective meat quality measures. 4. Both strains showed a diurnal rhythm with most birds observed outside around sunrise and before sunset. Feed and strain both affected the distribution of the birds on the outdoor area, with more birds on feed M than L observed outside, and with more LAB birds outside and using more of the outdoor area than ROSS. This was reflected in some of the carcase and meat quality measures. Feather pecking and cannibalism only occurred in LAB. 5. ROSS was found to have a faster growth rate, poorer litter quality, more dermal lesions and impaired mobility, reflected in low usage of outdoor area and poor gait score compared with LAB. 6. This together with the presence of pectoral myopathies make ROSS unsuitable for 12 week growth in free range production systems. The occurrence of feather pecking and cannibalism in LAB make this particular Labresse cross questionable for meat-type poultry production.

An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria.

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.

Staying good while playing god--the ethics of breeding farm animals.

Modern genetics has given us some very efficient tools with which to alter the characteristics of animals. To date, farm animal breeders have mainly used these tools to increase productivity. Thus, each new generation of farm animals matures faster, yields more milk, or produces more meat or eggs, than the previous one. Despite these apparent benefits, modern farm animal breeding has had severe negative consequences, including effects on the quality of the animals' lives and biodiversity. The aim of this paper is to discuss the goals and consequences of farm animal breeding within an ethical context. First, a description of what has happened to broilers and dairy cattle as breeders have pursued the goal of ever more efficient production is given. Second, the ethical values that ought to underlie future breeding schemes are discussed. It is suggested that there are in fact two very different ethical approaches: the 'quality of life-based' approach and the 'preservationist' approach. A view combining elements from both approaches is advanced. Finally, an example is given of how it is possible, in practice, to pursue an ethically defensible breeding goal without compromising production efficiency.

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane.

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic residue of leucine-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs. Thus, our observations suggest that the position, the exact sequence, and surrounding residues are major determinants of the function of leucine-based receptor sorting motifs.