Severe prothrombin deficiency caused by prothrombin-Edmonton (R-4Q) combined with a previously undetected deletion.

BACKGROUND: During infancy, a male patient experienced several life-threatening bleeding episodes. Standard coagulation tests revealed that the patient's plasma prothrombin activity was 8%, while his father's and mother's levels were 74% and 62%, respectively. OBJECTIVES: A molecular genetic approach was used to determine the molecular basis of prothrombin deficiency within the family. PATIENT/METHODS: Prothrombin genomic DNA fragments were amplified by using the polymerase chain reaction (PCR). In addition, liver cDNA fragments were amplified from the patient by using reverse transcription (RT) and PCR. The nucleotide sequences of the DNA fragments were determined. RESULTS: A novel, heterozygous point mutation (g.1755 G > A, named prothrombin-Edmonton) was detected in the patient and his mother, resulting in the mutation of Arg-4 in the prothrombin propeptide to Gln (R-4Q). RT-PCR analysis of the patient's liver sample demonstrated the presence of two mRNA transcripts that differed by the presence or absence of exon 11. Real-time PCR analysis on genomic DNA and cDNA confirmed a deletion (g.10435_10809del) in the paternal allele. CONCLUSIONS: The patient has a maternally-inherited point mutation (R-4Q) and a paternally-inherited deletion. By analogy with the previously reported factor IX San Dimas, the R-4Q mutation probably causes under-carboxylation of prothrombin and poor cleavage of the propeptide in the hepatocyte. The deletion probably results in a polypeptide that lacks 50 amino acids from the protease domain; this is likely to impair folding, secretion, stability and/or activity of the truncated prothrombin. The two mutations combine to give the prothrombin deficiency observed in the patient.

Presence of Ras guanyl nucleotide-releasing protein in striosomes of the mature and developing rat.

Ras signal transduction pathways have been implicated as key regulators in neuroplasticity and synaptic transmission in the brain. These pathways can be modulated by Ras guanyl nucleotide exchange factors, (GEF) which activate Ras proteins by catalysing the exchange of GDP for GTP. Ras guanyl nucleotide-releasing protein (RasGRP), a recently discovered Ras GEF, that links diacylglycerol and probably calcium to Ras signaling pathways, is expressed in brain as well as in T-cells. Here, we have used a highly selective monoclonal antibody against RasGRP to localize this protein within the striatum and related forebrain structures of developing and adult rats. RasGRP immunolabeling was found to be widespread in the mature and developing rat forebrain. Most notably, it presented a prominent patchy distribution throughout the striatum at birth and at all postnatal ages examined. These patches were found to correspond with the striosomal compartment of the striatum, as identified by micro-opioid receptor labeling in the adult. RasGRP-immunoreactivity was also observed in the matrix-like compartment surrounding these patches/striosomes but appeared later in development and was always weaker than in the patches. In both striatal compartments, RasGRP was exclusively expressed by medium-sized spiny neurons and showed no preference for neurons that project either directly or indirectly to the substantia nigra. At the ultrastructural level, immunogold labeling of RasGRP was confined to the cell bodies and dendritic shafts of these output neurons. We conclude that the prominent expression of RasGRP in striosomes may be of significance for diacylglycerol signaling in the striatum, and could be of importance for the processing of limbic-related activity within the basal ganglia.

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

Experience with aggressive therapy in three children with unresectable malignant liver tumors.

BACKGROUND: Children with malignant liver tumors often present with unresectable disease but need not be considered incurable. The advent of effective chemotherapy makes aggressive management feasible, as our experience with three such patients demonstrates. Procedure and Results One child with an unresectable undifferentiated sarcoma of the liver and two others with unresectable primary hepatoblastoma and lung metastases were treated with initial chemotherapy, followed by aggressive surgical management. Treatment with chemotherapy followed by hepatectomy and liver transplantation (cadaveric or live donor) in two children has resulted in disease-free survivals of 79 and 38 months. The third patient is alive and well 24 months following chemotherapy and aggressive resection of the primary and 12 metastatic lesions. CONCLUSIONS: Initial chemotherapy for unresectable liver tumors with or without metastases is supported by the review of the literature. Consideration of orthotopic liver transplantation (OLT) from cadaveric or living related donor is warranted when the malignancy is demonstrably chemosensitive, independent of initial staging. Aggressive resection of primary and metastatic disease may be called for in selected cases.

RasGRP is essential for mouse thymocyte differentiation and TCR signaling.

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes. Here we show that thymi of RasGRP-null mutant mice have approximately normal numbers of immature thymocytes but a marked deficiency of mature, single-positive (CD4+CD8- and CD4-CD8+) thymocytes. In Ras signaling and proliferation assays, mutant thymocytes showed a complete lack of response to DAG analogs or T cell receptor (TCR) stimulation by antibodies. Thus, TCR and DAG are linked through RasGRP to Ras signaling.

Organization and chromosomal locations of Rap1a/Krev sequences in the mouse.

The human RAP1A gene encodes a protein that apparently can antagonize the function of oncogenic ras genes in gene transfer experiments, but its normal function is unknown. To understand the function of this gene, we have undertaken a study of the mouse homolog, Rap1a. The complete coding sequence of a mouse Rap1a cDNA has been determined, and genomic clones representing three distinct Rap1a species were recovered. We find that Rap1a is located on distal mouse Chromosome (Chr) 3 near Nras, Ampd-1, Tshb, Ngfb, and Atp1a1. Two related sequences (Rap1a-rs1 and Rap1a-rs2) were also characterized. Rap1a-rs1, which was not localized, has a sequence very similar to the Rap1a cDNA, suggesting that it has been recently acquired by the mouse genome. Rap1a-rs2 is more distantly related to the gene sequence and is located on Chr 2 near Actc-1.

Stability of retrovirally transduced markers in a rat cell line.

A MoMLV-based retroviral vector capable of transmitting and expressing both the human hypoxanthine phosphoribosyltransferase (hprt) coding sequence and the Herpes simplex type 1 thymidine kinase (tk) gene has been constructed. After infection of a rat cell line, cell clones were selected on the basis of expressing both markers. They were subsequently found to contain a single provirus of the expected topology. The ease with which loss of expression of the markers can be monitored has allowed us to make observations on the stability of proviral genes. In particular, we have found indirect evidence of strong position effects on proviral gene expression by comparing the characteristic frequency of marker loss in different clonal proviral lines. Effects of the selection protocol on the apparent frequency of variants have also been noted. Finally, a combination of molecular and genetic observations lead us to invoke chromosome loss as the major factor influencing marker stability in this system.

The characterization of a mutant affecting DNA metabolism in the development of D. melanogaster.

Using a "single-fly" nucleic acid hybridization method, we have surveyed a collection of D. melanogaster strains in search of variants which affect DNA complementary to the polypyrimidine sequence corresponding to one strand of the 1.705 satellite. Hybridization of labelled polypyrimidine probe to polypurine sequence in nucleic acid extracts of single flies, followed by thermal chromatography over hydroxyapatite led to the identification of one variant. The strain Cy/M(2)S2(10) produced excess hybrid, much of which had low thermal stability. A developmental analysis of the low-melt hybrid phenotype showed that certain tissues, in particular the ovaries, were affected. In addition to the biochemical phenotype, the break down of nurse cell nuclei in Cy/M(2)S2(10) ovaries during oocyte maturation was abnormal. A genetic analysis demonstrated that both the biochemical and cytological phenotypes were the consequences of a single recessive mutation in the DNase-1 gene on chromosome III. Studies with purified DNA demonstrated that the low-melt hybrid phenotype resulted from the accumulation of low molecular weight DNA complementary to the polypyrimidine probe.

Chi activity during transduction-associated recombination.

Chi is a genetic element that stimulates phage lambda recombination by the Escherichia coli recBC pathway during lytic infection [Stahl, F. W. (1979) Annu. Rev. Genet. 13, 7--24]. Herein we show that chi in lambda prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation. This demonstration encourages the view that chi may influence genetic exchange in E. coli in the total absence of lambda.

Chi mutation in a transposon and the orientation-dependence of Chi phenotype.

Chi, an element that stimulates recombination via the E. coli RecBC pathway, can arise by spontaneous mutation in the transposon Tn5. When in phage lambda in one orientation, the mutant transposon confers Chi+ phenotype (large plaque and a high rate of exchange near the transposon). In the other orientation, however, the transposon does not confer Chi+ phenotype. The mobility of the transposon allows us to show that the Chi+ orientation of the mutant Tn5 is the same at different locations in lambda. These include a site near gene J, one in gam at 69, one to the right of gam at 73 and several to the right of R between 95.7 and 99.5. To the right of R, the mutant transposon could be found in only one orientation, that which confers Chi+ phenotype. We speculate that the other orientation of Tn5 in that locale is lethal to lambda. The orientation-dependence of Chi+ phenotype also revealed that Tn5 flip-flops in lambda.

Evolution of polypyrimidines in Drosophila.

We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, bu the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polpurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.--These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA . These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.