Defined Chain Length Research Articles

Fatty acyl chain composition in the determination of renal membrane order.

The relative roles of phospholipid fatty acyl chain length and phospholipid fatty acyl chain unsaturation in the determination of rat renal brush border membrane order were examined using multilamellar liposomes. Exposure of brush border membranes to sphingomyelinase resulted in a time- and concentration-dependent decrement in sphingomyelin content. Liposomes prepared from lipid extracts of these membranes were reconstituted to defined phosphatidylcholine (PC)/sphingomyelin (SPH) ratios with pure synthetic PCs of defined chain length and degrees of unsaturation. Mixed-acid PCs from bovine liver, egg, and the rat renal brush border membrane were also examined. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) at 37 degrees C was used to reflect acyl chain packing. The steady state anisotropy of DPH in liposomes isolated from the rat renal brush border membrane averaged 0.205 +/- 0.001, n = 8. When liposomes were reconstituted to PC/SPH ratios of 1.1, 1.6, and 2.4 with saturated PCs of acyl chain length 16 to 22, differences in anisotropy between groups were not observed. However, when PCs containing unsaturated or mixed-acid fatty acyl chains were introduced, anisotropy decreased in a concentration dependent fashion. These data suggest that phospholipid fatty acyl chain unsaturation, but not acyl chain length, has a powerful influence on renal brush border membrane order and the PC/SPH ratio is an important determinant of renal membrane order by virtue of the unsaturated fatty acids normally present with these phospholipids.

Die Struktur kollagenähnlicher Homo‐ und Heteropolytripeptide, 4. Polytripeptide durch repetitive Peptidsynthese und Verbrückung von Oligopeptiden

AbstractCollagen‐ like oligopeptides of defined chain length were synthesized via the following tripeptides which were combined stepwise: Boc‐Gly‐L‐Pro‐L‐Pro‐OH (1), Boc‐Gly‐L‐Pro‐L‐Lys‐OH (2), Boc‐L‐Ala‐Gly‐L‐Pro‐OH (4), Boc‐Gly‐L‐Pro‐L‐Leu‐OH (6), and Boc‐L‐Ala‐L‐Hyp(OBz)‐Gly‐OH (9). Two methods for covalent bridging of three oligopeptides of the same sequence were applied resulting in the formation of polypeptides of type 22 or 23.

Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.

Minimal requirements for in vitro activity of chemically defined synthetic antigens as immunogenic carrier molecules

A primary antibody response to 2,4-dinitrophenyl (Dnp)-oligolysines of defined chain length was induced in vitro. A molecular size of at least eight lysine residues was critical for the induction of antibody response to the hapten, but the location of the hapten on the carrier did not seem to play a significant role in this regard. Shorter peptides were nonimmunogenic, but did not paralyze the response to other, noncrossreacting antigens. The electric charge of the carrier molecule was found to have an effect on immunogenicity in vitro. A synthetic copolymer with a positive charge was immunogenic, whereas a similar molecule carrying a negative charge was inactive. On the other hand, changing the negative charge of carriers such as polyglutamic acid was not sufficient to render them immunogenic. Furthermore, neutralization of the negative charge of the surface of the spleen tissue by preincubation with positive polymers did not enhance the response to conjugates of the hapten with negatively charged carriers. These observations are interpreted on the basis of higher affinity of positively charged molecules for the negatively charged cell surface. Accordingly, the specific binding of the antigen to the cell surface is made more stable, and this ensures stimulation.

On the mechanism of oligonucleotide-primed DNA synthesis.

Oligodeoxyribonucleotides from various sources and of defined chain length were examined for their capacity to prime DNA synthesis on base complementary and non‐complementary single‐stranded DNA templates. Using Escherichia coli DNA polymerase I as enzyme, primer activity could be detected at chain lengths as few as five to six nucleotides. The activity was higher on base complementary templates and increased progressively with chain length from 0.07% priming efficiency to about 20% at chain length 16. On non‐complementary templates primer activity was low with maximal efficiencies around 0.1% at chain lengths of 7–10. Polypyrimidine nucleotides and homooligonucleotides of dT, dA and dC showed very low primer efficiencies (0.0001%–0.03%). The high priming activity of the complementary oligonucleotides strongly depended on the preincubation with the template and was paralleled by the stable annealing of primer molecules to the template DNA. It is concluded that the annealing reaction of template and primer is the rate‐limiting step in chain initiation and that the stability of the base‐paired primer template duplex determines the primer efficiency of an oligonucleotide.

Annealing Properties of Oligoribonucleotides Derived from T5 DNA-directed RNA Compared to Those from T2 and T7 DNA-directed RNAs

Abstract RNA made in vitro on a T5 phage DNA template was partially digested with ribonuclease T1 and fractionated according to chain length. The oligoribonucleotides of various chain lengths were tested for their annealing at different temperatures to denatured DNAs immobilized on membrane filters. As observed before for the T-even and other T-odd phage DNAs, a chain length of 10 or more ribonucleotides is required to form a ribonuclease-resistant complex with denatured T5 DNA in the absence of Mg2+. The shortest oligoribonucleotides capable of forming a complex with denatured T5 DNA display a high degree of specificity for species: these oligoribonucleotides do not anneal to either T2 or T7 DNA. As expected, the temperature for optimum complex formation and the melting temperature of the oligoribonucleotide-denatured DNA complex increase with the chain length of the oligoribonucleotide. With increasing chain length, the average base composition increasingly reflects the base composition of the parent RNAs. For the same chain length, T5-produced oligoribonucleotides having a G+C content intermediate between those of T2- and T7-produced oligoribonucleotides form complexes having stabilities intermediate between those of the latter. There are linear relationships between the melting temperatures and the G+C contents for oligoribonucleotides of a defined chain length. This makes it possible to characterize oligoribonucleotide-denatured DNA complexes as a function of both the chain length and the base composition of the oligoribonucleotide.

The enzymic hydroxylation of protocollagen models

1. Synthetic polymers of l-prolyl-l-prolylglycine of defined chain length, (Pro-Pro-Gly)(n), were found to be substrates for the enzyme protocollagen-proline hydroxylase, with optimum chain length n=5. Boiling the polymer (Pro-Pro-Gly)(15) increased its activity as a substrate but had no effect on (Pro-Pro-Gly)(5). 2. Protection of both or one of the N- and C-terminal groups made (Pro-Pro-Gly)(3) a better substrate, and collagenase digestion of hydroxylated tert.-pentyloxy-carbonyl-(Pro-Pro-Gly)(3) benzyl ester indicated that the central prolyl residues were the major points of hydroxylation. 3. The results suggest that the long-chain peptides are optimum substrates but that a triple-stranded structure is inhibitory for hydroxylation.

Étude du RNA synthétisé in vitro

Studies on RNA synthesized in vitro The bulk of RNA synthesized by purified E. coli RNA polymerase on calf thymus and T 7 DNA as templates, has been chromatographed on methylated serum albumin column. The elution pattern is very similar to those obtained with pulse labelled RNA from cells. The RNA's obtained in vitro are eluted, as three main fractions, namely II, III and IV, located respectively before the 16-S and 23-S RNA and after the 23-S RNA. RNA's from Fractions II and III are not aggregates made of either different RNA chains or RNA and carrier rRNA, as demonstrated by the following data: (1) Fractions II and III are present even if the carriers rRNA are omitted. (2) Thermal or mechanical degradation of the RNA lowers Fraction IV, increases Fractions II and III and induces the formation of new peaks. (3) The elongation of RNA can be limited by incorporation of cordycepin triphosphate. Under these conditions, Fraction IV disappears and Fractions II and III lose their sharpness. (4) The RNA length can be directly measured by the ratio 3H belonging to the chain nucleotides, to 32P attached to the initiator nucleotide. The evolution of this ratio through the pattern is coherent and the relation between the position of a given RNA in the elution pattern and its length, is perfectly reproducible. After 20 min synthesis, the lengths of RNA's constitutive of Fractions II, III and IV, are respectively: 1350–1500, 2700–2900 and 5000–5500 nucleotides. When the synthesis of RNA is allowed to proceed after a short pulse with a labelled nucleotide followed by a chase, the evolution of the pattern of fractions shows that the RNA of Fractions II and III are precursors of the RNA IV which are also growing. In fact, when a synchronized elongation of the RNA chains is allowed to proceed, we obtain only Fraction IV. These data, toegether with the fact that no nuclease activity has ever been detected in our preparations, show that the particular heterogeneity of the RNA is a genuine property of the material synthesized in vitro. The relative quantities of Fractions II, III and IV vary with the DNA, RNA polymerase and nucleotide concentrations. The kinetics of appearance of the different fractions and the knowledge of their respective lengths show that the average rate of RNA synthesis decreases with time of incubation. It is 90–100 nucleotides/sec during the first 30 sec and 20–27 nucleotides/sec during the first 3 min. One explains the presence of these specific intermediary fractions by the fact that the rate of synthesis is not homogeneous along the entire DNA molecule, and that the initiation of RNA chains is not instantaneous. The kinetics of the synchronized elongation of the RNA chains shows 4 plateaux during the first 3 min, even though each plateau does not represent an intermediary fraction of defined chain length. It appears nevertheless that two kinds of phenomena, one rapid, one slow, are involved in the elongation of the RNA chains.