Randy L. Allen

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.

Micronuclei isolated from growing cells of Tetrahymena thermophila contain three H1-like polypeptides alpha, beta, and gamma. Micronuclei isolated from young conjugating cells (3-7 h) also contain a larger molecular weight polypeptide, X, which is being actively synthesized and deposited into these nuclei (Allis, C. D., and J. C. Wiggins, 1984, Dev. Biol., 101:282-294). Pulse-chase experiments (with growing and conjugating cells) suggested that X is a precursor to alpha and that alpha is further processed to gamma and a previously undescribed and relatively minor species, delta. These precursor-product relationships were supported by cross-reactivity with polyclonal antibodies raised against alpha and peptide mapping. While beta consistently became labeled under chase conditions (both in growing and mating cells), it was not clear whether it is part of the vivo processing event(s) which interrelates X, alpha, gamma, and delta. Beta was not recognized by alpha antibodies. Despite this uncertainty, these results suggest that proteolytic processing serves to generate significant changes in the complement of H1-like histones present in this nucleus.