Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice

Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.     

Genomic Circuitry Underlying Immunological Response to Pediatric Acute Respiratory Infection

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SPATIAL AND TEMPORAL VARIATION IN THE SEED BANK OF A SEMIARID GRASSLAND

The spatial and temporal variability in the seed bank of a semiarid grassland in Colorado was evaluated using soil cores. Spatial variability in the soil storage of germinable seeds was assessed by sampling two shortgrass plant communities on sites with the same climatic conditions but differing in soil texture. Differences between communities were largely the result of annual plant seeds. Eight sampling dates over two years were used to assess temporal variability, which was more important to the storage of germinable seeds than spatial variability. Differences in the numbers of seeds stored were found between the two sampling years, and seasonally within years. The number of seedlings that emerged from the samples ranged from 122–2,748/m². A poor correspondence was found between the species composition of the plant communities and the storage of germinable seeds; however, the species composition of the seeds produced on the sites tended to have a high similarity with the seedlings that emerged. Most of the species had a transient rather than a persistent presence in the seed bank.     

Utilization of Dissolved Nitrogen by Heterotrophic Bacterioplankton: Effect of Substrate C/N Ratio

The significance of dissolved combined amino acids (DCAA), dissolved free amino acids (DFAA), and dissolved DNA (D-DNA) as sources of C and N for marine bacteria in batch cultures with variable substrate C/N ratios was studied. Glucose, ammonium, alanine, and phosphate were added to the cultures to produce C/N ratios of 5, 10, and 15 and to ensure that phosphorus was not limiting. Maximum bacterial particulate organic carbon production (after 25 h of incubation) was inversely correlated with the C/N ratio: with the addition of identical amounts of carbon, the levels of production were 9.0-, 10.0-, and 11.1-fold higher at C/N ratios of 15, 10, and 5, respectively, relative to an unamended control. The bacterial growth efficiency increased from 22% (control cultures) to 44 to 53% in the cultures with manipulated C/N ratios (C/N-manipulated cultures). Net carbon incorporation from DCAA, DFAA, and D-DNA supported on average 19, 4, and 3% (control cultures and cultures to which only phosphate was added [+P cultures]) and 5, 4, and 0.3% of the particulate organic carbon production (C/N-manipulated cultures), respectively. In the C/N-manipulated cultures, a 2.6- to 3.4-fold-higher level of incorporation of DCAA, relative to that in the control cultures, occurred. Incorporation of D-DNA increased with the substrate C/N ratio, suggesting that D-DNA mainly was a source of N to the bacteria. Organic N (DCAA, DFAA, and D-DNA) sustained 14 to 49% of the net bacterial N production. NH₄⁺ was the dominant N source and constituted 55 to 99% of the total N uptake. NO₃^- contributed up to 23% to the total N uptake but was released in two cultures. The studied N compounds sustained nearly all of the bacterial N demand. Our results show that the C/N ratio of dissolved organic matter available to bacteria has a significant influence on the incorporation of individual compounds like DCAA and D-DNA.     

Effectiveness of intensive insulin therapy by multiple daily injections and continuous subcutaneous infusion: a comparison study in type 2 diabetes with conventional insulin regimen failure.

OBJECTIVE: To compare the effectiveness of two intensified insulin regimens, i.e., pump delivery versus multiple daily injections in patients with type 2 diabetes not optimally controlled with conventional insulin therapy. RESEARCH DESIGN AND METHODS: Seventeen type 2 diabetes patients uncontrolled by two daily injections of regular plus NPH were randomly assigned in a cross-over fashion to either three daily injections of lispro plus NPH or pump device delivering lispro. HbA1c, 6 points capillary blood glucose, 24-hour continuous glucose monitoring system tracings and global satisfaction score were evaluated at the end of each 12-week treatment period. RESULTS: HbA1c decreased from 9.0+/-1.6% to 8.6+/-1.6% with multiple injections and 7.7+/-0.8% with pump device (p<0.03). Capillary blood glucose was lowered at all time-points with pump, but only at morning with multiple injections (p<0.01). Compared to conventional therapy, pump reduced hyperglycemic area under curve by 73% (p<0.01), but multiple injections by only 32% (p=0.08). Rate of hypoglycemia was not increased and patient's satisfaction was comparable with both intensive treatments. CONCLUSIONS: Pump therapy provides a better metabolic control than injection regimens, and seems to be safe and convenient in patients with type 2 diabetes who fail to respond to conventional insulin therapy.     

In Vivo Inactivation of Lysozyme by Ozone

Bronchial mucus contains large quantities of the antibacterial enzyme lysozyme. Acutely exposing mice or rabbits to ozone reduced the amount of active lysozyme obtainable by bronchopulmonary lavage. The effect was proportional to ozone concentration, as well as to duration of exposure. Enzyme activity returned to normal levels during the 12 hr following exposure. Ozone (5 muliters/liter; 5 parts per million) for 3 hr reduced lysozyme levels approximately 30%. Studies of the release of lysozyme by alveolar cells support the theory that loss of activity is due to in vivo oxidation of lysozyme.     

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.     

Highly preferred targets for retrovirus integration

A central feature of retrovirus replication is integration of the provirus into host cell DNA, but the specificity of this step for cell target sequences has not been clarified. To investigate this issue, we developed a method for screening and comparing large numbers of unselected integration events. Using a replication-competent Rous sarcoma virus containing a bacterial suppressor tRNA gene as a selectable marker, we obtained collections of clones comprising integrated provirus together with host flanking sequences. Hybridization and sequence analysis of the flanking sequence reveals the presence of a number of strongly preferred integration targets. Within these targets, independent integration events occur at sites identical to the base.