Fetal Sheep Liver Research Articles

Induction of cytosolic phosphoenolpyruvate carboxykinase in the ovine fetal liver by chronic fetal hypoglycemia and hypoinsulinemia.

Fetal glucose production has been observed in the chronically hypoglycemic, hypoinsulinemic (HH) fetal lamb. The purpose of this study was to test the hypothesis that induction of hepatic gluconeogenic enzymes occurs in this condition. The activities of both mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase, and glucose-6-phosphatase, three key enzymes of gluconeogenesis, were determined in fetal sheep liver from HH lambs and controls (CONT). Pregnant ewes were maintained chronically hypoglycemic by continuous hyperinsulinemic clamps from approximately 80 d of gestational age (53% of gestation) for 6 wk. Fetuses (gestational age: HH = 136 +/- 2.6, CONT = 133 +/- 3.7 d) were maintained chronically hypoglycemic [HH = 0.51 +/- 0.05 versus CONT = 1.22 +/- 0.11 mmol/L (9.2 +/- 1.0 versus 21.9 +/- 11.9 mg/dL)] and hypoinsulinemic (HH = 3.3 +/- 0.6 versus CONT = 12.0 +/- 2.2 microU/mL) and delivered by cesarean section after measurement of fetal glucose production rate. Hepatic cytosolic PEPCK was 6.0 +/- 1.4 nmol/min/mg protein in CONT and 19.7 +/- 2.5 in HH lamb (p < 0.05), whereas mitochondrial PEPCK was not different between the two groups. Neither fructose-1,6-diphosphatase or glucose-6-phosphatase activities nor plasma glucagon levels were different between groups. These results suggest that chronic fetal hypoglycemia and hypoinsulinemia prematurely induce hepatic cytosolic PEPCK in the fetal lamb. The observed fetal glucose production in the HH fetal lamb may be due to gluconeogenesis.

The placental lactogen receptor in maternal and fetal sheep liver: regulation by glucose and role in the pathogenesis of fasting during pregnancy.

To clarify the roles of glucose and insulin in the regulation of the PL receptor in fetal and maternal sheep liver, we administered iv glucose to pregnant ewes during a 72-h fast. The binding of ovine PL (oPL) to hepatic membranes from glucose-infused ewes and their fetuses was compared with the binding of oPL to tissues of fasted, saline-infused sheep and sheep fed normally ad libitum. Fasting of pregnant ewes caused a 58-70% reduction in the number of PL receptors in fetal and maternal liver. Intravenous administration of glucose during fasting increased the number of PL receptors in fetal liver by 137.4%. In contrast, glucose administration during fasting had no effect on the number of PL receptors in maternal liver. The number of PL binding sites in fetal liver correlated positively with fetal weight (r = 0.59) and length (r = 0.54) and with fetal plasma glucose (r = 0.69) and insulin (r = 0.55) concentrations. In contrast, PL binding was inversely related to fetal plasma oPL concentrations (r = -0.70). These findings suggested that glucose, insulin, and/or oPL may regulate PL binding in the ovine fetus. To determine whether glucose or insulin exert direct effects on the PL receptor in ovine fetal tissues, we examined the binding of radiolabeled oPL to ovine fetal hepatocytes and fibroblasts in culture. The specific binding of oPL to fetal hepatocytes was low and variable (1.0 +/- 0.5%) and it was not possible to assess reliably the effects of glucose or insulin supplementation. The specific binding of oPL to fetal fibroblasts (5.4 +/- 0.6%/mg) was unaffected by variations in media glucose concentrations (5.5-16.5 mM) or by pretreatment with insulin (10-1000 ng/ml). The results of these studies demonstrate that glucose and other nutritional factors regulate the expression of the PL receptor in fetal and maternal sheep liver. Alterations in PL binding play roles in the metabolic adaptation of the mother and fetus to nutritional deprivation and stress.

The placental lactogen receptor in maternal and fetal sheep liver: regulation by glucose and role in the pathogenesis of fasting during pregnancy

The placental lactogen receptor in maternal and fetal sheep liver: regulation by glucose and role in the pathogenesis of fasting during pregnancy

Negative Regulation of Angiotensinogen Gene Expression by Glucocorticoids in Fetal Sheep Liver

The effect of glucocorticoids in regulating liver angiotensinogen gene expression was studied in chronically instrumented fetal sheep during the last trimester of gestation and was compared with the expression of other hepatic genes (prothrombin, factor IX, and albumin). Four sets of twins were studied at 118 d of gestation, and three sets were studied at 138 d of gestation (term, 145 d). One of each set of twins was infused intraperitoneally with cortisol (5 mumol.mL-1.h-1) for 48 h, whereas the other twin received the same volume (1 mL/h) of normal saline. Plasma cortisol concentration increased from 0.32 +/- 0.12 and 2.7 +/- 0.12 nmol/100 mL to 44.2 +/- 20.0 and 37.7 +/- 8.2 nmol/100 mL in 118- and 138-d fetuses, respectively, during the cortisol infusion; no changes were observed in fetuses infused with saline alone. At the end of the infusion period, the animals were anesthetized, the fetal liver was removed, and total cellular RNA was isolated and probed for angiotensinogen, prothrombin, factor IX, and albumin. The results demonstrated that cortisol infusion decreased angiotensinogen mRNA by 61% in 138-d fetuses and albumin mRNA expression by 2.4-fold in 118-d fetuses and by 3.4-fold in 138-d fetuses. On the other hand, cortisol had no effect on fetal factor IX gene expression but increased prothrombin mRNA levels by 65% in 118-d fetuses and 62% in 138-d fetuses. Taken together, our results suggest that, during fetal life, angiotensinogen gene expression is negatively regulated by glucocorticoids. This effect is not universal because cortisol increases fetal prothrombin gene expression.

Release of glucose from the liver of fetal and postnatal sheep by portal vein infusion of catecholamines or glucagon.

1. The blood flow to the liver in fetuses near term, newborn and adult sheep was measured by the Fick principle, using radionuclide-labelled plastic microspheres, before and during infusion of adrenaline, noradrenaline or glucagon. 2. Glucose output and lactate consumption by the liver in sheep of each age group were calculated by application of the Fick principle using the concentration gradients of these metabolites measured in blood samples obtained, simultaneously with blood flow measurements, from catheters chronically implanted in the inflow and outflow vessels of the liver. 3. Catecholamines were infused into the portal vein of fetuses near term at a rate comparable with that at which they are known to be secreted in the sheep fetus during moderate to severe hypoxia. The cardiovascular and metabolic responses to these infusions were found to be comparable with those that occur in the fetus during hypoxia. 4. Catecholamines increased glucose output from the liver in all except the immediate post-partum animals. Catecholamines were less effective than glucagon in promoting glucose release. The mean increments in glucose output during adrenaline infusion were 0.055 +/- 0.015 mmol min-1 (100 g liver)-1 in the fetus, 0.122 +/- 0.024 mmol min-1 (100 g)-1 in the 2-week-old lambs, 0.078 +/- 0.019 mmol min-1 (100 g)-1 in young lambs and 0.049 +/- 0.012 mmol min-1 (100 g)-1 in the adult sheep. During glucagon infusion the mean glucose output increments were 0.146 +/- 0.023 mmol min-1 (100 g)-1 in the fetus, 0.274 +/- 0.085 mmol min-1 (100 g)-1 in the 2-week-old and young lambs and 0.180 +/- 0.054 mmol min-1 (100 g)-1 in the adult. Adrenaline was more potent than noradrenaline, suggesting that the major glycogenolytic response might be beta-receptor mediated. 5. In the immediate newborn period the output of glucose from the liver was high (0.20 +/- 0.05 mmol min-1 (100 g liver)-1 and was not statistically significantly increased by infusion either of glucagon or of catecholamines which resulted in similar increments of glucose output of about 0.128 +/- 0.133 mmol min-1 (100 g)-1. It is probable that the high output of glucose reflected the high endogenous circulating levels of catecholamines and glucagon in these animals at birth and that further infusions failed to add significantly to the already near-maximal glucose release.(ABSTRACT TRUNCATED AT 400 WORDS)

The nucleotide and deduced amino acid sequences of insulin-like growth factor II cDNAs from adult bovine and fetal sheep liver

The nucleotide and deduced amino acid sequences of insulin-like growth factor II cDNAs from adult bovine and fetal sheep liver

Endocrine Regulation of Tissue Glucose-6-Phosphatase Activity in the Fetal Sheep during Late Gestation*

Endocrine regulation of tissue glucose-6-phosphatase activity in utero was examined by measuring enzyme levels in liver and kidneys of fetal sheep during the second half of gestation and after experimental manipulation of fetal plasma cortisol and insulin levels. Tissue glucose-6-phosphatase activities increased toward term in parallel with the rise in fetal plasma cortisol. At birth, the activities were significantly higher than in utero, but significantly less than in adult nonpregnant sheep. Fetal hypophysectomy lowered fetal plasma cortisol and reduced hepatic and renal glucose-6-phosphatase activities compared with those in intact fetuses near term. Conversely, intrafetal cortisol infusion raised fetal plasma cortisol and significantly increased tissue glucose-6-phosphatase activity to values similar to those in older fetuses. When the data from these groups of fetuses and the newborn lambs were combined, there was a significant positive correlation between the plasma cortisol level and the glucose-6-phosphatase activity in both liver and kidney. Fetal hypoinsulinemia was induced by fasting the ewe for 48 h and by fetal pancreatectomy. Fetal hepatic and renal glucose-6-phosphatase activities were higher in fasted than in fed animals, while pancreatectomy had little apparent effect on enzyme activity in either tissue. However, when differences in plasma cortisol were taken into account, hepatic, but not renal, glucose-6-phosphatase activities were higher in both groups of hypoinsulinemic fetuses than would have been observed in normoinsulinemic animals with a similar plasma cortisol level. Partial correlation analysis of the data showed that plasma insulin and cortisol were both significant influences on hepatic glucose-6-phosphatase activity in utero, but plasma cortisol had the more pronounced effect. Cortisol, therefore, appears to be a physiological regulator of tissue glucose-6-phosphatase activity in utero and enhances the glucogenic capacity of the sheep fetus during late gestation.

Placental lactogen receptors in maternal sheep liver: effects of fasting and refeeding

In a recent study we demonstrated that fasting of the pregnant ewe reduces the number of placental lactogen (PL) receptors in fetal sheep liver. In the present study we examined the effects of a 72-h fast on the number and affinity of PL receptors in maternal sheep liver. Fasting caused a 57% reduction in the number of hepatic ovine PL receptors; this effect was reversed by refeeding. There were no changes in the affinity of the PL receptor, the subunit structure of the receptor, or the extent of occupancy of the receptor by endogenous circulating maternal hormones. The number of hepatic PL receptors was correlated positively with the maternal plasma concentrations of glucose and insulin, suggesting that these factors may regulate PL binding to maternal tissues during pregnancy. In addition, PL receptor number was correlated positively with maternal plasma insulin-like growth factor I (IGF-I) concentrations, suggesting that a reduction in hepatic ovine PL binding may contribute to the reduction in maternal IGF-I during fasting. Fasting produced a 72% reduction in the number of ovine growth hormone receptors in maternal liver and an 83% increase in hepatic insulin binding. These findings indicate that fasting of the pregnant ewe reduces the number of PL receptors in maternal and fetal liver. The reduction in PL binding may contribute to maternal and fetal hyposomatomedinemia and may play a role in the pathogenesis of the intrauterine growth retardation that accompanies maternal nutritional deprivation.

Purification of a distinct placental lactogen receptor, a new member of the growth hormone/prolactin receptor family.

Recent findings from this laboratory suggest that the biological actions of placental lactogen (PL) in mammalian fetal tissues are mediated through binding of the hormone to a distinct and unique PL receptor. We have now purified this receptor from fetal and maternal sheep liver, characterized its binding to PL, growth hormone (GH), and prolactin (PRL), and determined its molecular weight by SDS-PAGE and by affinity cross-linking techniques. Soluble extracts containing specific, high-affinity (Kd 0.5 nM) PL binding activity were prepared by incubating ovine fetal and maternal liver microsomes with 1% Triton X-100. The detergent solubilized PL receptor was purified two- to threefold by ion-exchange chromatography and an additional twofold by gel exclusion chromatography on Sepharose 6B. The PL receptor was then purified 75,000- to 125,000-fold by affinity chromatography using a column of ovine PL (oPL) coupled to Affi-Gel 10. The molecular weight of the oPL receptor as determined by SDS-PAGE and by cross-linking techniques was 44,000 +/- 2,000 (range 40,000-48,000). The purified receptor bound 125I-oPL specifically and with high affinity (Kd 0.5 nM) but did not bind either radiolabeled ovine GH or ovine PRL. In addition, in competition studies using 125I-oPL as the radioligand, the purified PL receptor bound unlabeled oPL with a potency 30-50 times greater than that of ovine GH and 500-1,000 times greater than that of ovine PRL. These findings demonstrate the presence of a specific PL receptor in fetal and maternal sheep liver. The PL receptor, together with the GH and PRL receptors, constitute a family of distinct but related hormone receptors that differ in their relative affinities for PL, GH, and PRL. Changes in the expression of the three receptors may mediate changes in the hormonal control of growth during the transition from fetal to postnatal life.

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BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4–5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minima1 changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-l-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth.

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.

Effect of a Maternal Fast on the Urea Cycle Enzymes of the Ovine Fetus

SummaryActivities of five urea cycle enzymes were measured in maternal and fetal sheep liver during the normal fed state and following 5 days of fasting. Six ewes and 10 fetuses were studied in both the fed and fasted periods at 132 days gestation (term: 147 days) for liver protein and enzyme levels. Results indicated that protein content increased during fasting in both the maternal and fetal liver. Fetal liver weight was decreased during fasting from 108 ± 8.5 to 71 ± 8.2 g (mean ± SD) (p &lt; 0.001). Fed state fetal enzyme activities per gram liver were 50–125% of maternal values. After fasting, four of the five fetal enzymes increased approximately twofold to fivefold (per gram tissue) (ornithine transcarbamylase did not change). Only one enzyme (argininosuccinase) increased significantly in maternal liver. Total liver activities gave similar results. These data indicate that the in vivo studies that demonstrate a doubling in fetal urea production in the fasted sheep in later gestation are associated with parallel increases in the fetal hepatic activities of several enzymes that are responsible for urea synthesis.

Effect of a maternal fast on the urea cycle enzymes of the ovine fetus.

Activities of five urea cycle enzymes were measured in maternal and fetal sheep liver during the normal fed state and following 5 days of fasting. Six ewes and 10 fetuses were studied in both the fed and fasted periods at 132 days gestation (term: 147 days) for liver protein and enzyme levels. Results indicated that protein content increased during fasting in both the maternal and fetal liver. Fetal liver weight was decreased during fasting from 108 +/- 8.5 to 71 +/- 8.2 g (mean +/- SD) (p less than 0.001). Fed state fetal enzyme activities per gram liver were 50-125% of maternal values. After fasting, four of the five fetal enzymes increased approximately twofold to fivefold (per gram tissue) (ornithine transcarbamylase did not change). Only one enzyme (argininosuccinase) increased significantly in maternal liver. Total liver activities gave similar results. These data indicate that the in vivo studies that demonstrate a doubling in fetal urea production in the fasted sheep in later gestation are associated with parallel increases in the fetal hepatic activities of several enzymes that are responsible for urea synthesis.

1227 UREA CYCLE ENZYMES DURING FASTING IN THE OVINE FETUS

Activities of five urea cycle enzymes were measured in maternal and fetal sheep liver during the normal fed state and following five days of fasting. Six ewes and 10 fetuses were studied in both the fed and fasted periods at 132 days gestation (term = 147 days) for liver protein, DNA and enzyme levels. Results indicated that protein and DNA contents remained unchanged during fasting in both the maternal and fetal liver. Fetal liver weight was decreased during fasting from 108 ± 23 to 71 ± 21 gm (mean ± SD). Fed state fetal enzyme levels were at 25-50% of maternal values per mg DNA and 50-125% of maternal levels per gm liver. After fasting, four of the five enzymes increased by 2.3 to sixfold (per mg DNA) in fetal liver; only one enzyme (AS) increased significantly in maternal liver. Activities of each enzyme per whole fetal liver'gave similar results.These data indicate that the in vivo studies which demonstrate a doubling in fetal urea production in the fasted sheep in latter gestation are associated with parallel increases in the fetal hepatic activities of several enzymes responsible for urea synthesis.

1228 LACTATE IS A GLYCOGENIC PRECURSOR IN THE OVINE FETUS

Glucose (Glu) levels are exceedingly low in the ovine fetus and the ungulate liver has little measurable glucokinase. Nonetheless, at term, the fetal (Fet) sheep liver contains significant glycogen stores. Recent suggestions that glycogenesis in the adult may proceed through the gluconeogenic pathway led us to examine the capacity of lactate (Lac) to serve as a glycogenic precursor in the chronically catheterized Fet sheep. Six ovine fetuses were prepared with aortic (Ao) and inferior vena cava (IVC) catheters. On day 5 post surgery, after a maternal (Mat) 48 hour fast, a steady state Mat venous infusion of Glu was begun sufficient to raise Mat blood Glu from 19.41.6 mg/dl to 48.6±6.2 mg/dl. A simultaneous infusion of 300 uC (U-14C) Lac was administered to the Fet IVC over a 5 hour period to steady state. Six Fet Ao and Mat art. samples were obtained q 10 min during the last 60 min of the infusion. The fetus and ewe were then sacrificed and liver removed for determination of glycogen and 14C glycogen enrichment. Minimal radiolabel was found in Mat blood or liver glycogen. Fet blood levels were Lac 13.6±1.3 mg/dl (SA 42±9 uc/mg×103), fructose 70.2±10.4 mg/dl (SA 1.0±.2 uc/mg×103), Glu 23.2±4.3 mg/dl. No Lac-derived labelled Fet Glu could be reliably detected. Fet hepatic glycogen was 40.0±6.5 mg/g liver (SA 1.4±.5 uc/mg×103). The SA of glycogen and absent Glu labelling suggests that Lac is a precursor of Fet glycogen through the gluconeogenic path. This is teleologically important, since placental conversion of Mat Glu to Lac maintains higher levels of Lac in the Fet circulation and seems to be a means for Fet conservation of energy stores.

Application of reversed-phase high-performance liquid chromatography using pre-column derivatization with o-phthaldialdehyde for the quantitative analysis of amino acids in adult and fetal sheep plasma, animal feeds and tissues

The concentration of amino acids in adult and fetal sheep plasma, liver, muscle and animal feeds was determined by reversed-phase high-performance liquid chromatography using an automated data acquisition system and pre-column o-phthaldialdehyde derivatization. Of the seventeen amino acids in the standard mixture, fifteen including glycine and threonine were completely resolved in a total analysis time of 50 min. The clear resolution, high degree of precision and accuracy, relatively rapid analysis and the lack of interference from chemical contaminants in feed and tissue hydrolysates render this technique suitable for routine analysis of large numbers of biological samples of nutritional interest.

Alterations in hepatic monodeiodination of iodothyronines in the diabetic rat

Alterations in hepatic monodeiodination of iodothyronines in the diabetic rat

Hemoglobin switching in sheep: characteristics of BFU-E-derived colonies from fetal liver

Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8–256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to &gt; 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

Hemoglobin Switching in Sheep: Characteristics of BFU-E-Derived Colonies From Fetal Liver

Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8-256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to > 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen.

DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.