The timing of proanthocyanidin (PA) biosynthesis in leaves of
Onobrychis viciifolia (sainfoin) was examined using
nucleotide probes, enzyme assays and immunochemical detection. To achieve
this, a chalcone synthase (CHS) cDNA clone was isolated
and a PCR-amplified DNA fragment corresponding to part of a dihydroflavonol
reductase (DFR) gene was obtained from sainfoin. The
high sequence homology of the CHS clone, both at the
nucleotide and amino acid levels, with CHS sequences from other species,
confirmed the identity of the clone. The identity of the PCR fragment was
confirmed by sequence homology to known DFR sequences.
The level of DFR mRNA, DFR and leucoanthocyanidin
reductase (LAR) enzyme activities, CHS mRNA and CHS
protein were at a maximum in young, unexpanded leaves of sainfoin. The levels
of DFR mRNA, and DFR and LAR enzyme activity declined
rapidly and were absent in older tissues. PA content of leaves declined
slightly on a fresh and dry weight basis in the more mature phases of leaf
development; however, PA content on a per leaf basis continued to increase
throughout development. The apparent anomaly between the activity of critical
enzymes (LAR and DFR) and the accumulation of PA indicates that the process of
proanthocyanidin biosynthesis is still poorly understood.
Analysis of the tammar wallaby beta-lactoglobulin cDNA and inferred amino acid sequences reveal extensive sequence divergence from the eutherian beta-lactoglobulins. Conserved residues include the cysteines and a number of individual amino acids involved in structure and ligand-binding. The only region of extended similarity is a heptapeptide sequence which may impart specificity to its interaction with a receptor protein. Northern analysis of total mammary RNA revealed two transcripts which result from differential utilization of polyadenylation signals. The concentration of beta-lactoglobulin mRNA increased in late lactation and correlates with increases in milk production and levels of milk fat. Quantification of beta-lactoglobulin mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the gene is dependent on prolactin alone and that expression is not modulated by other hormones known to play a role in the initiation of lactation in eutherians.
Lotus corniculatus L. plants were transformed with
Agrobacterium rhizogenes binary vector carrying the
maize Sn regulatory gene driven by the 35S promoter.
These plants showed modifications in the pattern of accumulation of
proanthocyanidin (PA). All the transformed plants but one showed an increase
in PA content in the root relative to control untransformed and control
gus gene transformed plants (C). With respect to the
PAaccumulation in leaves, Sn transgenic plants were
grouped in two classes: suppressed (S), that showed a consistent reduction of
foliar PAcontent, and unsuppressed (U) that did not differ significantly from
controls. Dihydroflavanol reductase (DFR) and leucocyanidin reductase (LAR)
enzyme activities in S and U plant leaves mirrored the changes seen with
foliar PA accumulation. LAR activity in the roots was consistent with the root
PA levels. Mature Sn mRNA accumulated in the leaves of U
plants, but not in leaves of S plants; however, leaves of both S and U plants
were able to initiate Sn transcription. All
Sn-transformed plants accumulated
Sn message in root tissue. A possible negative
interaction of Sn and an unidentified homologous
endogene is proposed for explaining the behaviour of S plants.
The gene for alpha-lactalbumin has been cloned from a tammar wallaby (Macropus eugenii) mammary gland cDNA library. Tammar alpha-lactalbumin has approximately 50 and 30% homology to the alpha-lactalbumins of eutherians at the levels of nucleotide and protein sequence respectively. Comparison of the inferred tammar polypeptide sequence with the sequence of the eutherian proteins reveals extensive divergence at almost all of the non-essential amino acid residues. However, the hydropathy plots of the tammar protein are almost identical to those of eutherian alpha-lactalbumins, suggesting that protein conformation is conserved. The tammar gene encodes a transcript of approximately 975 bases. Northern blot analysis of hormone-stimulated mammary gland explants shows that maximal induction of alpha-lactalbumin mRNA is dependent on prolactin and that expression is not modulated by other hormones that play a role in the initiation of lactation in eutherians.
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