No abstract

A cDNA library from lamprey liver was constructed in pBR322 and screened with a synthetic mixed oligonucleotide probe, the sequence of which was based on a partial amino acid sequence of the lamprey fibrinogen gamma chain determined by conventional procedures. Among the positive clones was one containing a 600-base insert that covered the carboxy-terminal third of the chain and another with a 1950-base insert that stretched more than full length. The two inserts were sequenced by the Maxam-Gilbert procedure. The DNA sequencing was corroborated by reference to the amino acid sequences of five cyanogen bromide peptides that compose the carboxy-terminal 130 amino acids, as well as to a number of tryptic peptides from elsewhere in the molecule. The clone with the smaller insert (6G) contained 594 nucleotides (not counting G and C tails), 435 of which are coding and correspond to residues 264-408 of the gamma chain. The remaining 159 nucleotides included the terminator codon followed by a noncoding segment. The larger clone (2E) coded for 408 amino acids that could be readily aligned with the 411-residue human gamma chain. A 24-residue signal peptide adjacent to the proposed amino terminal was also inferred. The amino acid sequence of the fibrinogen gamma chain has been differentially conserved during evolution, the lamprey and human sequences being more than 70% identical in certain key regions but dropping to less than 25% in other sections, including the segment thought to be a part of the "coiled coils". Overall, the resemblance amounts to 50% identity. Of the 10 cysteines found in mammalian chains, 9 are at identical positions, but the tenth, which in mammalian fibrinogens is a part of the interdimeric bridging, is absent in the lamprey.

This ambulatory nursing intensity tool proved to be adaptable to heterogeneous patient populations.

The influence of alkaline earth metal ions, alkali metal ions, ammonium ions, and polyamines upon the equilibria between high-salt-washed wheat germ ribosomes and their subunits was investigated by light scattering. The Mgz+-dependent, reversible association of ribosomal subunits was studied with buffers containing 100 mM concentrations of the chloride salts of various monovalent cations. These studies indicate that K+ and Na+ are most effective in dissociating the ribosomes into subunits, followed by Rb+ and then NH4+. The dissociation curves determined with buffers containing K+, Na', Rb', and NH4+ ions exhibit [Mg"] 1/2 values of 1.4, 1.4, 1.2, and 0.7 mM, respectively. The divalent cations, in general, strongly promote subunit association but differ in their abilities to do so. Ca2+ associates the ribosomal subunits more effectively than Sr2+, which is more effective than Mgz+. In the presence of 100 mM KCl and 0.15 mM MgClz, Ca2+-, Sr2+-, and Mgz+-dependent association curvesx e subunits of both eucaryotic and procaryotic ribosomes are capable of reversible association and dissociation. The types and concentrations of various cations present play a major role in determining the position of the equilibrium between the intact ribosome and its subunits. Kinetic, thermodynamic, and ion-specificity studies have been performed with Escherichia coli ribosomes in an effort to explain the roles of MgZ+, K+, and polyamines in the association of the subunits. Conflicting mechanisms have been proposed as a result of these studies. One model suggests that Mgz+ binds to specific sites on the subunits and thereby promotes subunit association (Petermann, 1964;Goldberg, 1966). This model is supported by the data of Zitomer & Flab (1972), who found that MgZ+, Ca2+, and polyamines promote ribosomal subunit association while K+ promotes subunit dissociation. Ca2+, however, was less effective than Mg2+ in promoting subunit association, suggesting that the ions were acting at specific sites. In contrast, the electrostatic model suggests that Mg2+ neutralizes the charged groups on the ribosomal subunits. This reduction in the electrostatic repulsion between the subunits would allow association. The studies of Walters & Van Os (1970, 1971) on yeast ribosomes and Wishnia & Boussert (1977) on E . coli ribosomes support the electrostatic model. Calculations by these groups also showed that the reduction of the electrostatic repulsion by Mg2+ theoretically is sufficient to promote subunit association. Nieuwenhuysen et al. (1980), using Artemia salina ribosomes, have shown that the molar volume changes produced by ribosomal subunit association are similar to those observed with E. coli type A ribosomes. Furthermore, they have suggested that this large volume change is consistent with two From the exhibit [M2+lIl2 values of 0.9, 1.1, and 1.4 mM, respectively. The abilities of the polyamines to associate ribosomal subunits in buffers containing 100 mM KCl and 0.20 mM MgClz were investigated. Both spermine ( [~p m ]~/~ = 0.09 mM) and sp...

The effects of various cations and cosolvents on the conformation of wheat germ ribosomes and ribosomal subunits have been investigated by using the techniques of circular dichroism and differential hydrogen exchange. A class of hydrogens on both the 40S and 60S subunits exchange out more rapidly as the Mg2+ concentration is raised, indicating that Mg2+ causes a change in subunit conformation. Ca2+ and the polyamines produce a similar increase in the rate of hydrogen exchange. These results suggest that increases in cation concentrations permit a tightening of ribosome structure and a greater degree of internalization of the rRNA. The cosolvent glycerol causes an alteration in the CD spectrum of 80S ribosomes in both the nucleic acid and protein portions of the spectrum. Glycerol also causes a decrease in the rate of exchange of a number of hydrogens on both the 40S and 60S subunits. These results are interpreted to mean that glycerol favors a more native, less denatured structure in the ribosome.