Nuclear translocation of a maternal CCAAT factor at the start of gastrulation activates Xenopus GATA-2 transcription.

The transcription factor GATA-2 is present in blood cell precursors and plays a pivotal role in the control of erythroid differentiation. In Xenopus embryos, low levels of GATA-2 mRNA are maternally derived, while the onset of zygotic GATA-2 expression coincides with commitment to haematopoietic lineages. However, its initial transcriptional activation is not restricted to the presumptive blood islands, but occurs throughout ventral and lateral regions, in all three germ layers. In order to determine how this expression pattern is controlled, we have isolated and characterized the Xenopus GATA-2 gene. We show that 1.65 kb of 5' flanking sequences are sufficient to direct both correct transcriptional initiation in oocytes and appropriate temporal and spatial gene expression in early embryos. The transgene is activated during gastrulation and by neurula stages in predominantly expressed in the ventral hemisphere. We demonstrate that a CCAAT element is necessary for gene activity in both systems and that extracts prepared from oocytes and embryos contain a factor which specifically recognizes this element. We also show that cytoplasmic localization inhibits the function of this CCAAT factor until the beginning of gastrulation, when the zygotic GATA-2 gene is activated. These observations extend our understanding of the mechanisms by which maternal factors control the temporal activation of transcription in early vertebrate embryos.

Use of dpn I restriction enzyme to assess newly replicated gene copies in amplifiable vector systems.

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5 , this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3 ) or calcium phosphate mediated gene transfer (see Chapter 2 ) (3-5). At least some of the DNA introduced into the cells enters into the nucleus, where it is thought to be assembled into chromatin (6), and is maintained extrachromosomally for at least 48 h. During this time, the cellular chromosomal DNA may have undergone one or more rounds of DNA replication. However, the extrachromosomal transfected DNA will not replicate unless the DNA sequences contained in the plasmid include a DNA origin of replication recognized by the host cell. Origin sequences have so far proved difficult to identify in eukaryotic chromosomes. In contrast, viral genomes, such as SV40 and polyoma, have well-characterized origins of replication (7), which, when included on DNA.

Role for DNA replication in beta-globin gene activation.

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

5' structural motifs and Xenopus beta globin gene activation.

We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.

Simian virus 40-mediated cis induction of the Xenopus beta-globin DNase I hypersensitive site.

Regions in chromatin which are hypersensitive to the action of DNase I appear to be associated with sites of genetic activity; the association between DNase I hypersensitivity and transcriptional activation is well known. In the case of the chicken beta-globin gene the establishment of a DNase I hypersensitive site is dependent on tissue-specific trans-acting factors. Such factors have also been implicated in the action of viral and cellular enhancers, which are themselves hypersensitive to DNase I. Enhancers have been defined operationally as DNA sequences which act in cis to potentiate transcription from their own, heterologous or cryptic promoters. This activity is essentially unaffected by changes in the orientation, position (5' or 3') or distance of the enhancer element with respect to its cognate promoter. We demonstrate here that the transcriptional rescue of the Xenopus laevis beta-globin gene by simian virus 40 (SV40) sequences including the enhancer coincides with the conferment of DNase I hypersensitivity upon that gene, and that this occurs in the absence of any change in the complement of trans-acting factors. These results suggest that a propensity to form sites hypersensitive to the action of DNase I is encoded in the primary sequence of DNA, and that this predilection is aggravated by SV40 sequences, perhaps through a mechanism dependent on supercoiling.

The efficiency of utilization of metabolizable energy and apparent absorption of amino acids in sheep given spring- and autumn-harvested dried grass.

Three experiments were conducted with sheep given spring-harvested dried grass (SHG) and autumn-harvested dried grass (AHG). The first was a calorimetric trial to determine the metabolizable energy (ME) content of each grass and the efficiency with which sheep utilize their extra ME intakes above the maintenance level of intake. The second examined the relative amounts of extra non-ammonia-nitrogen (NAN) and individual amino acids absorbed from the small intestine per unit extra ME intake as the level of feeding was raised from energy equilibrium (M) to approximately 1.5 M. The third was a further calorimetric trial to investigate the effect of an abomasal infusion of 30 g casein/d on the efficiency of utilization of AHG. The ME content of the SHG (11.8 MJ/kg dry matter (DM] was higher than that of AHG (10.0 MJ/kg DM). The efficiency of utilization of ME for productive purposes (i.e. above the M level of intake; kf) was higher when given SHG (kf 0.54 between M and 2 M) than when given AHG (kf 0.43 between M and 2 M). As the level of intake of each grass was raised from M to 1.5 M there was a greater increment in the amounts of NAN (P less than 0.001) and the total amino acid (P less than 0.05) absorbed from the small intestines when sheep were given the SHG (NAN absorption, SHG 5.4 g/d, AHG 1.5 g/d, SED 0.54; total amino acid absorption SHG 31.5 g/d, AHG 14.3 g/d, SED 5.24). Infusion of 30 g casein/d per abomasum of sheep given AHG at M and 1.5 M levels of intake increased (P less than 0.05) the efficiency of utilization of the herbage from kf 0.45 to kf 0.57. Consideration is given to the possibility that the higher efficiency of utilization of ME in sheep given SHG may be related to the amounts of extra glucogenic amino acids absorbed from the small intestine which provide extra reducing equivalents (NADPH) and glycerol phosphate necessary for the conversion of acetate into fatty acids.

Effect of kidney bean (Phaseolus vulgaris) toxin on tissue weight and composition and some metabolic functions of rats.

Inclusion of raw kidney bean (Phaseolus vulgaris) proteins in the diet for rats was shown to affect the weight of some internal organs. Of these, in addition to the well-known hypertrophy of the pancreas attributable to dietary trypsin inhibitors, the observed atrophy of the thymus and the doubling in weight of the small intestine are related to the protein or lectin content of the bean diet, or both. Changes in tissue composition of the small intestine were also recorded. Its protein content increased by about 40-50% and carbohydrate content doubled suggesting the occurrence of increased mucinous glycoprotein secretion. Increased DNA content (by about 30-40%) however also indicated mucosal hyperplasia. Changes were also observed in mineral content, urea concentration and some enzyme activities in sera and urine, possibly as a result of disturbances in systemic metabolism or hormone levels, or both. The results gave further support to previous suggestions that the oral toxicity of kidney-bean lectins involves local reactions in the small intestine in combination with their effects on the systemic immune system and general metabolism.

Calcium and phosphorus absorption in lambs exposed to Trichostrongylus colubriformis.

Ten lambs (29 +/- 1.2 kg) reared parasite-free and prepared with rumen, duodenal and ileal cannulae were paired and one of each pair was given a daily oral dose of 2500 Trichostrongylus colubriformis larvae for 14 weeks. Untreated animals received the amount of ration consumed by their infected pair-mates the previous day. During weeks 6 and 12 of infection, all lambs underwent a 7 day calcium (Ca) and phosphorus (P) balance. During weeks 7 and 13, duodenal and ileal samples were collected to determine the amounts of Ca and P entering and leaving the small intestine. The infection caused varying degrees of feed refusal in all infected animals. As a result, the data on Ca and P in excreta and the amounts of Ca and P entering and leaving the small intestine were regressed against dry matter (DM) intake for each group at each period. There were no between-period differences in these relationships. Calcium absorption and retention were unaffected by the stress of infection. Infection affected several aspects of P metabolism. Blood P concentrations were markedly reduced. Absorption of P from the small intestine was greater (P less than 0.01) in control lambs (at 1 kg DM intake 6.6 g per day) than in infected animals (2.2 g P per day), but there was a greater (P less than 0.05) duodenal flow rate of P in control lambs which suggested much higher rates of salivary secretion of P than in infected animals. Phosphorus flow rates at the ileum were greater (P less than 0.01) in infected lambs, despite the lower duodenal flow rates, which indicated a major abnormality (P less than 0.01) in small intestine absorption of P in infected animals; this may have contributed to the growth check experienced by these lambs.

The relation between food intake and abomasal emptying and small intestinal transit time in sheep.

The relation between the level of food intake and gastrointestinal motility and digesta flow in the abomasum and small intestine was studied in sheep fitted with nichrome-wire electrodes in the gut wall, an abomasal and a duodenal catheter and a terminal ileal cannula. Abomasal volume and outflow were calculated from CrEDTA dilution in six sheep and small intestinal transit time by the passage of Phenol Red in ten sheep. The frequency of the migrating myoelectric complex of the small intestine was not altered by the level of food intake but the duration of the periods of irregular spiking activity, the amplitude of abomasal activity and the frequency of duodenal rushes were decreased as the level of food intake was decreased. There was a linear relation between the level of food intake (FI) and abomasal outflow (mean with SEM: 327 (69) ml/h for each kg FI/d; P less than 0.01), and abomasal volume (mean with SEM: 344 (50) ml/kg FI per d; P less than 0.001), without any significant change in the half-time of marker dilution in the abomasum. Small intestinal transit time decreased with an increase in food intake (mean with SEM: -54.9 (5.6) min/kg FI per d; P less than 0.001). It is concluded that abomasal volume and the rate of digesta flow from the abomasum and along the small intestine are linearly related to the level of food intake.

The organization of the tadpole and adult alpha globin genes of Xenopus laevis.

Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.