Analysis of von Willebrand factor mRNA from the lung of pigs with severe
von Willebrand disease by using a human cDNA probe
QY Wu, BR Bahnak, L Coulombel, D Kerbiriou-Nabias, L Drouet, G Pietu, P Meulien, A Pavirani, JP Caen and D Meyer
Institut National de la Sante et de la Recherche Medicale (INSERM) U.143,
Hopital de Bicetre, Le Kremlin-Bicetre, France.
To examine the control of porcine von Willebrand factor (vWF) biosynthesis
we cloned human vWF complementary DNA (cDNA) and investigated the
expression of the vWF gene in lungs from normal pigs and pigs with severe
von Willebrand's disease (vWD). Recombinant clones spanning approximately
90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA
library by screening with oligonucleotides. One clone spanning nucleotides
960 to 3,240 of human vWF cDNA was used to investigate the steady-state
levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically
determined to be homozygous for vWD. This clone hybridized with genomic DNA
from pig leukocytes when Southern blots were processed under very stringent
conditions; therefore, human cDNA clones were considered valid probes to
detect porcine mRNA. Northern blot analysis of total RNA from normal pig
lung and human umbilical vein endothelial cells identified the vWF mRNA as
a molecular species of approximately 9.0 kilobases (kb). A very faint to
undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a
decreased rate of transcription of the vWF gene. Sucrose density gradient
centrifugation of RNA from the vWD pigs confirmed by Northern analysis that
the high-molecular weight fractions contained vWF mRNA and at the same size
as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA
processed under stringent conditions demonstrated that the relative ratio
of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the
ratio observed in normal pigs. Because the amount of vWF mRNA is not
correlated to the amount of vWF activity or antigen in plasma of vWD pigs
we conclude that posttranscriptional events are also probably involved in
abnormal expression of vWF in these animals.
Volume 71,
Issue 5,
pp. 1341-1346,
05/01/1988
Copyright © 1988 by The American Society of Hematology
