46,XX gonadal dysgenesis, short stature, and recurrent metabolic acidosis in two sisters.

Gonadal (ovarian) dysgenesis in 46,XX individuals is genetically heterogeneous. We report on two sisters who, in addition to primary ovarian failure, have marked short stature and recurrent episodes of dehydration with metabolic acidosis. Studies performed during one of these episodes suggested mitochondrial dysfunction; however, results of biochemical analysis of electron transport chain activity in skeletal muscle and mitochondrial DNA studies were normal. We discuss the phenotype in relation to previously described conditions of 46,XX gonadal dysgenesis. We suggest this constellation of findings represents a new syndrome.

Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center.

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns.

Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.

Matrix-attachment regions in the mouse chromosome 7F imprinted domain.

We have mapped the matrix-attachment regions (MARs) in 200 kilobases of the mouse Chromosome (Chr) 7F imprinted domain. MARs are genetic elements known to have effects in cis on methylation at nonimprinted loci. The imprinting of the Igf2 and Ins2 genes is dependent on the transcription of the downstream H19 gene. The transcription of H19 is dependent in turn on its methylation status. The cis-acting regulators of methylation at this site are not known. As MARs are potential regulators not only of methylation but also other elements of genomic imprinting, we mapped the MARs within the 200 kilobases around H19. This report describes the mapping of four MARs from this region.

Physical linkage of two mammalian imprinted genes, H19 and insulin-like growth factor 2.

Parental imprinting is a phenomenon in mammals whereby the maternal and paternal alleles of a gene are differentially expressed. Three murine genes have been shown to display this type of allele-specific expression. Two of them, insulin-like growth factor-2 (Igf-2) and H19, map to the distal end of mouse chromosome 7, but are imprinted in opposite directions. Pulsed-field gel electrophoresis and large-fragment DNA cloning were utilized to establish a physical map that includes H19 and Igf-2. Igf-2 lies approximately 90 kilobases of DNA 5' to H19, in the same transcriptional orientation. This physical proximity is conserved in humans, based on pulsed-field gel analysis. We conclude that H19 and Igf-2 constitute an imprinted domain.

Parental imprinting of the mouse H19 gene.

THE mouse H19 gene encodes one of the most abundant RNAs in the developing mouse embryo. It is expressed at the blastocyst stage of development, and accumulates to high levels in tissues of endodermal and mesodermal origin (H. Kim, unpublished result). After birth the gene is expressed in all tissues except skeletal muscle. It lacks a common open reading frame in the 2.5-kilobase RNA, but has considerable nucleotide sequence similarity between the genes of rodents and humans. Expression of the gene in transgenic mice results in late prenatal lethality, suggesting that the dosage of its gene product is strictly controlled. The H19 gene maps to the distal segment of mouse chromosome 7, in a region that is parentally imprinted, a process by which genes are differentially expressed on the maternal and paternal chromosomes. We have now used an RNase protection assay that can distinguish between H19 alleles in four subspecies of Mus, to demonstrate that the H19 gene is parentally imprinted, with the active copy derived from the mother. This assay will be of general use in assaying allele-specific gene expression.

Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations.

It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene. Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts. Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid. Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations. We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase. We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation.

Characterization and distribution of angiotensin-II receptors in the primate fetus.

The binding characteristics and distribution of angiotensin-II (AII) receptors were studied in Cynomolgus monkey fetuses and one second trimester human fetus. In contrast to the adult monkey, in which binding was confined to the adrenal gland, kidney, and smooth muscle, autoradiographic studies in the monkey fetus revealed the presence of high density binding in mesenchymal tissue throughout the body, especially in skeletal muscle and dermis. In the kidney at 11 weeks, binding was mainly associated with connective tissue surrounding primitive nephrons, while at 17 weeks, binding distribution was similar to that in the adult primate kidney, being confined to the glomeruli and smooth muscle of blood vessels, with low binding in the tubules. In fetal monkey adrenal, binding was high in the medulla and connective tissue of the capsule, and low in the zona glomerulosa, while in the adult, binding was high in the zona glomerulosa and medulla. In membrane preparations from fetal monkey skin and skeletal muscle, binding was specific for AII analogs, but in contrast to the adult adrenal, it was not affected by guanyl nucleotides. Scatchard analysis showed a single class of sites with a Kd of 0.6 +/- 0.1 nM and a capacity of 3060 +/- 8.3 fmol/mg, higher than that of the adult adrenal glomerulosa (605 +/- 30 fmol/mg). Specific binding for AII analogs was also present in human fetal skin and skeletal muscle membranes, where Scatchard analysis indicated a Kd of 0.8 nM and a binding capacity of 640 fmol/mg. The transient expression of abundant AII receptors during the phase of rapid growth in the fetus in conjunction with the known effects of AII on cellular growth suggest a role for AII during fetal development in the primate.

Novel sites of expression of functional angiotensin II receptors in the late gestation fetus.

In the adult, the peptide hormone angiotensin II (AII) is primarily known as a regulator of circulatory homeostasis, but recent evidence also suggests a role in cell growth. This study of AII in late gestation rat fetuses revealed the unexpected presence of receptors in skeletal muscle and connective tissue, in addition to those in recognized adult target tissues. The AII receptors in this novel location decreased by 80 percent 1 day after birth and were almost undetectable in the adult. Studies in fetal skin fibroblasts showed that the receptors were coupled to phospholipid breakdown, with concomitant increases in inositol phosphate and cytosolic calcium. The abundance, timing of expression, and unique localization of functional AII receptors in the fetus suggest a role for AII in fetal development.

Distribution of angiotensin II receptors and renin in the mouse fetus.

To investigate the ontogeny of the renin-angiotensin system we studied the characteristics and location of angiotensin II (AII) receptors in mouse fetuses and examined sites of renin mRNA expression by in situ hybridization and Northern blot analysis. Autoradiographic analysis of the binding of 125I-[Sar1,Ala8]AII to slide-mounted frozen sections of 17-day-old DBA/2N mice revealed abundant AII receptors widely distributed throughout the body. High receptor density was found in primitive mesenchymal tissue under the epidermis and surrounding muscle and cartilage, in skeletal and smooth muscle, and in all layers of the adrenal cortex. Lower receptor density was seen in the kidney, liver, and lungs. The autoradiographic staining was abolished by incubation of the sections with excess unlabeled AII. Scatchard analysis of the binding of 125I-[Sar1,Ala8,]AII to membrane-rich fractions of eviscerated fetuses showed a single type of high affinity receptors with a Kd of 2.9 x 10(-9) M and a receptor concentration of 3300 fmol/mg protein. Localization of renin mRNA was analyzed by in situ hybridization using an antisense 35S-labeled riboprobe transcribed from a mouse renin2 cDNA clone. Hybridization to fetal tissue sections showed high intensity staining in the kidney and adrenal cortex. Northern blot analysis confirmed the high expression of renin mRNA in the fetal kidney. The presence of an active renin-angiotensin system in the fetus was confirmed by the demonstration of renin-like activity and bioactive AII in fetal extracts. The widespread distribution of AII receptors in the fetus, compared to the discrete localization to specialized tissues in the adult, may indicate a unique role for the peptide during development.

Nonimmune hydrops fetalis in association with hemangioma of the umbilical cord.

Nonimmune hydrops fetalis is becoming the predominant form of fetal hydrops due to the declining frequency of Rh isoimmunization. Reported is the preterm delivery of a hydropic twin with umbilical cord and cutaneous hemangiomata. The unusual umbilical angiomatous malformation was associated with marked edema of the cord. This produced an ultrasonographic abnormality detected antenatally as a multicystic mass in close approximation to the fetal abdomen. The hydropic twin responded to aggressive neonatal management. It appears that hemangiomata of the umbilical cord may be causally related to fetal hydrops and may represent another entry in the differential diagnosis of this disorder.