mania parasites invade macrophages and survive 
within their hostile oxidative environment. 
One important parasite constituent in both the 
entry and survival process is a protease called glyco- 
protein 63 (gp63)- This is the most abundant pro- 
tein on the surface of stationary-phase promasti- 
gotes, which can invade macrophages, but is nearly 
absent from log-phase cells, which do not infect 
macrophages. Although log-phase promastigotes 
possess very little gp63, they have as much steady- 
state RNA for gp63 as do stationary promastigotes. 
Characterization of gp63 cDNAs from log- and 
stationary-phase promastigotes revealed three dis- 
tinct transcripts for gp63, each of which encodes a 
different form of the protease. One transcript of 2.7 
kb occurs in log cells; another of 3.0 kb, in station- 
ary cells; and a third of 3 .1 kb, a transcript of low 
abundance, is constitutively expressed. Cells that 
are intermediate between log and stationary phase 
contain all three transcripts. Each of the transcripts, 
which differ primarily in the length and sequences 
of their 3'-nontranslated regions, directed the syn- 
thesis of gp63 in a heterologous in vitro translation 
system, suggesting that mechanisms other than tran- 
scription regulate the gp63 levels. 
The gp63 RNAs are products of two clusters of 
genes. Nuclear run-on experiments showed that 
transcription initiation from all three gene clusters 
occurs continuously and that the steady-state levels 
of the three transcripts are regulated by post- 
transcriptional events. These post-translational regu- 
latory processes occur, at least in part, when a leader 
of 39 nucleotides is spliced onto the 5' ends of the 
gp63 transcripts. In addition, DNA transfections of 
plasmid constructs containing the different 3'- 
untranslated regions downstream of a luciferase 
gene suggest that the unique 3' nontranslated se- 
quences also influence the stabilities of the tran- 
scripts at different promastigote growth stages. 
Other experiments are in progress to examine the 
effect of deleting or altering the gp63 genes on the 
infectivity and survival of the leishmania parasite in 
macrophages. Although not completed, the work 
demonstrates that the regulation of gp63 levels in 
leishmania promastigotes occurs at several steps. 
Parts of this research are supported by a grant from 
the National Institutes of Health. 
Dr. Donelson is also Distinguished Professor of 
Biochemistry at the University of Iowa College of 
Medicine, Iowa City. 
Articles 
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