Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3' end formation

We previously demonstrated that a critical 35 bp region 3' of the AAUAAA is required for rabbit beta-globin mRNA 3' end formation. Recently, we synthesized and tested sequence elements derived from this region. Here, we report that a GU-rich and a U-rich sequence element are both required for efficient rabbit beta-globin mRNA 3' end formation. The efficiency of processing is restored to the wild-type level when the two elements are placed together and is greatly diminished when only one element is present. The level of 3' end formation is also decreased when the distance between the two elements is expanded. These results demonstrate that the GU-rich and U-rich elements function synergistically to restore efficient mRNA 3' end formation and that they most likely form a single requisite sequence 3' of the AAUAAA. Furthermore, we show that the effect of the GU-rich and U-rich sequence elements is position-dependent.     

Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39.

We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.     

Immunoaffinity fractionation of high-density lipoprotein subclasses 2 and 3 using anti-apolipoprotein A-I and A-II immunosorbent gels

High-density lipoprotein (HDL) subclasses 2 and 3 prepared by density gradient ultracentrifugation have been further fractionated by immunoaffinity chromatography using antibody affinity gels targetting the major HDL apolipoproteins, A-I and A-II. Fractions containing A-I without A-II (AI w/o AII) and A-I with A-II (AI w AII) were isolated from both density ranges. Whereas there were similar concentrations of the major subfraction (HDL3(AI w AII] in both males and females, the remaining subfractions were present in higher concentrations in females as compared to males, in the order HDL3 (AI w/o AII) less than HDL2(AI w AII) less than HDL2(AI w/o AII). The difference was most marked for HDL2 (AI w/o AII), where plasma concentrations in females were almost 3-fold greater than in males. Compositional analyses indicated that the plasma concentrations of the fractions, rather than their compositions, were the major determinants of male-female differences in HDL levels. In contrast, fractions defined by similar apolipoprotein criteria and isolated from different density subclasses (i.e., HDL2(AI w/o AII) vs. HDL3(AI w/o AII) and HDL2(AI w AII) vs. HDL3(AI w AII] showed major compositional differences. This is suggestive of distinct lipoprotein particles.     

Enzyme-assisted semisynthesis of polypeptide active esters and their use.

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.     

Tripartite sequences within and 3'' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human alpha-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human alpha-globin gene. This provides evidence for the role of poly(A) in the stability of mRNA.     

Acute Paracetamol Poisoning

Of 41 cases of acute paracetamol poisoning one died of gastrointestinal haemorrhage and acute massive necrosis of the liver, three became jaundiced, and 13 others had biochemical evidence of hepatocellular damage. Liver damage is a toxic effect which is present in most patients who ingest more than 15 g. of paracetamol. One patient with liver damage survived renal failure due to acute tubular necrosis. It is suggested that the renal lesion was also the result of paracetamol overdosage.Profound hypoglycaemia and metabolic acidosis may also complicate severe poisoning. Plasma levels of para-aminophenol fall rapidly, and procedures currently used to enhance the elimination of the drug cannot be expected to prevent development of hepatic damage.     

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.