Purification and structural properties of the precursors of the globin messenger RNAs

X-ray diffraction data have been obtained from sodium and calcium salts of a proteoglycan rich in chondroitin 4-sulfate isolated from the Swarm rat chondrosarcoma. When sodium is the only countercation associated with the proteoglycan, the oriented polysaccharide chains adopt a 3-fold helical conformation in the solid state and pack in a trigonal unit cell with dimensions a = b = 1.45 nm and c = 2.88 nm. Addition of small amounts of calcium or full conversion of the polyanion from a sodium to a calcium salt form results in a conformational transition to a somewhat more extended 2-fold structure.For the calcium salt X-ray intensity data were used to refine the polysaccharide conformation and packing arrangement in the unit cell. Two antiparallel chains were found to crystallize in an orthorhombic unit cell with space group P22₁2₁ and dimensions a = 0.745 nm, b = 1.781 nm and c = 1.964 nm. The individual helix axes intersect the base plane of the unit cell at (x_f = 0, y_f = 0) and ($\text{x}{}_{\mathrm{f}}\text{= 0, y}{}_{\mathrm{f}}\text{=}\text{1}\text{2}$), and the polyanions are crystallographically equivalent, being related by the symmetry of the space group.The conformation of chondroitin 4-sulfate is stabilized intramolecularly by O.3 … O.5 hydrogen bonds across the β(1 → 4) linkage as well as by OSO^?₃ … Ca²⁺ … ^?OOC co-ordination across the β(1 → 3) linkage. Within the lattice adjacent parallel chains interact through COO^? … Ca²⁺ … ^?OOC bridges, and each calcium co-ordination shell is completed with an additional five water molecules to form a distorted, square antiprism. These water molecules are hydrogen-bonded to neighboring polyanions, and all intermolecular interactions involve water bridges or calcium ion co-ordination.On the basis of the refined packing model and the known structural features of the proteoglycan, models are considered for proteoglycan organization in connective tissue. Consideration of the conformational directing influence and relative abundance of calcium in the intercellular matrix suggest that the secondary structure of chondroitin 4-sulfate in vivo is likely to be similar to the conformation described in this study.     

Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation.     

Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

Changes in haemopoietic sites during the metamorphosis of the lampreys Lampetra fluviatilis and Lampetra planeri

An investigation has been made in both the parasitic lamprey Lampetra fluviatilis and in its non-parasitic derivative Lampetra planeri of the rate at which the fat column replaces the typhlosole and nephric fold as the principal site of haemopoiesis. In the typhlosole, blood cell formation started to decline prior to the onset of external metamorphosis and had ceased within four weeks of the commencement of transformation. In the nephric fold haemopoiesis continued for several weeks in the region where the larval opisthonephros persisted but was never observed in the newly developing adult kidney. Soon after the onset of external metamorphosis the fat column started to become haemopoietic and later became the main site of blood cell formation. The rate at which the haemopoietic function was transferred from the nephric fold and typhlosole to the fat column was greater in L. fluviatilis than in L. planeri. Since a similar more rapid change in L. fluviatilis has also been found in the switch from larval to adult haemoglobin, the former type of haemoglobins may be produced only in erythrocytes originating in the nephric fold and typhlosole, whereas the latter type may be restricted to cells developed in the fat column. It is also suggested that the functional significance of the alteration in haemopoietic sites is related to changes at metamorphosis in the three regions where blood cell formation occurs.     

Multiple leader RNAs and messenger RNAs are transcribed from the la crosse virus small genome segment

Nucleotide sequencing has demonstrated that the Small genome segment of Bunyaviruses contains the genetic information for two viral proteins (N and NS_s) in overlapping reading frames (Akashi and Bishop, 1983; Cabradilla et al., in press). Using 3′ end-labeled genome probes, La Crosse virus (LAC) infected cells were shown to contain three leader RNAs, which start at position 1 and terminate at approximate positions 74, 95, and 115 from the 3′ end of the genome. Primer extension and S1 mapping studies have shown that LAC-infected cells contain seven mRNAs, five of which initiate at positions 1, 74, 101, 117, and 123 and two more which initiate at approximate positions 147 and 159 from the 3′ end of the Small genome segment. The existence of multiple leader RNAs and mRNAs from the Small genome segment may allow access to both overlapping reading frames by ribosomes that still initiate only at 5′ proximal AUGs, without the need to splice the mRNA.     

T7 early messenger RNAs are the direct products of ribonuclease III cleavage

T7 early RNAs were synthesized in vitro by transcribing T7 DNA with Escherichia coli RNA polymerase and treating the resulting precursor molecule with ribonuelease III. Oligonucleotide fragments from the 5′ and 3′ termini of several of the cleaved species were then selectively isolated. Structural analysis revealed sequences identical to the corresponding in vivo RNAs. Thus, the T7 early RNAs found in phage-infected cells appear to be the direct products of RNAase III cleavage of a large precursor molecule. We conclude further that RNAase III action on this particular natural substrate is a sequence-specific event.     

The morphology of amphibian skin vascularization before and after metamorphosis

Summary The skin vascularization was investigated troughout the ontogenetic development and in adults of two anurans, Rana temporaria and Bufo bufo, and two urodeles, Triturus vulgaris and Triturus cristatus. It was found that, contrary to the urodele larvae, the anuran tadpoles have a very sparse skin vascularization. During the early stages of anuran metamorphosis the skin capillary network becomes dense; later, during skin metamorphosis, a second, venous, network is formed as anastomoses between the subcutaneous vein ramifications. In the urodeles, metamorphosis is not accompanied by any significant morphological changes in the skin vascularization, and a subcutaneous network is not formed. It is suggested that the reduced skin vascularization in anuran tadpoles is an advanced larval character relative to the rich vascularization of the skin in urodele larvae. It is further suggested that anuran tadpoles have a reduced ability to utilize gaseous exchange through the skin. The function of the subcutaneous venous network found in anurans after metamorphosis is obscure; experiments indicate a vasomotor regulation which is neither adrenergic nor cholinergic.Abbreviations a arteriole - A artery - an anastomosis between the subepidermal capillary network and the subcutaneous venous network - C stratum compactum - E epidermis - ec subepidermal capillaries - S stratum spongiosum - sv subcutaneous venous network - v venule - V vein - vc venae comitantes