MITIFICACIÓN Y DESMITIFICACIÓN DE DON JUAN

Co-translatio nal

cleavage

Proprotein

_{Post-translational}

PA1

MW= 1 1 000

cleavage

Mature protei n

PA1 a

MW=6000

Mature protei n

PA1 b

MW=4000

68

The 2S seed storage proteins from castor bean (Sharief and Li, 1982), rapeseed (Ericson et aI., 1986), mustard seed (Menendaz-Arias, 1 988) and brazil nut (Ampe et aI., 1986) all show similarities to SF8, particularly in the position and number of cysteine residues (Kont

et al., 1990). There is very little experimental data available concerning SF8 although, a partial cDNA clone coding for SF8 has been constructed (Lilley et aI., 1 989) and was used

in this thesis to construct a chimeric gene for the expression of SF8 in the leaves of transgenic tobacco.

1.3.4 Protein Degradation in the Rumen.

The degradation of plant protein in the rumen follows the sequence;

Protein > Large Polypeptides > Small Polypeptides > Amino Acids > Ammonia

The proteolytic activity in the rumen is almost entirely associated with bacterial cells, with cell free rumen fluid and protozoa having very little proteolytic activity (Nugent and Mangan, 198 1). Nugent et al. (1983) suggested that the rate of proteolysis of a protein in the rumen was a function of;

( 1 ) Total protein concentration in the rumen, with a higher concentration favouring faster proteolysis.

(2) The degree of competitive inhibition of bacterial proteases by proteins resistant to degradation.

Nugent and Mangan (1981) suggested the production of NH3 from amino acids was comparatively fast and that the rate limiting step in protein degradation was the initial formation of large polypeptides. Degradation of protein to peptides and amino acids in the rumen is catalysed by proteases and peptidases which are located on the surface of the bacteria (Blackburn, 1968). However, initial breakdown of protein in the rumen is

correlated with protein solubility, as proteins must be in solution for bacterial proteases to work efficiently (Hungate, 1 966).

1.3.4.1 Degradation of High Sulphur Proteins and Casein in the Rumen.

There is very little experimental data available examining the rumen degradation of proteins containing a high proportion of SAA. However, in an in vitro rumen assay using rumen fluid from a sheep fed chaffed lucerne hay, ovalbumin, PAl (Spencer et al., 1988)

69

incubation, whilst casein (Spencer et al., 1988) was undetectable after 1 hour of incubation. Mangan ( 1 972) reported that the half-life of casein in the rumen of cows eating a

hay/concentrate diet was in the range of 5.6-12.3 min whilst ovalbumin had a half-life of 1 80 min. Mangan ( 1 972) suggested it was unlikely that any casein would have survived rumen degradation and flowed intact, into the abomasum, whilst ovalbumin was likely to flow undegraded into the abomasum. Therefore, transgenic plants expressing ovalbumin have more potential for improving animal production than transgenic plants expressing casein.

1.3.4.2. Degradation of Individual Amino Acids.

Varvikko ( 1986) reported that residues of barley and barley-straw remaining in nylon bags after suspension of the nylon bags in the rumen of a cow eating a grass silage/hay diet (4600gDM/d), had a higher EAA:NEAA ratio than prior to suspension in the rumen. The slower degradation of EAA compared to NEAA appeared to be related to the source of the protein, as nylon bag residues from rapeseed meal and ryegrass had unchanged

EAA:NEAA ratios. Branched-chain amino acids tended to increase in all feed residues, suggesting they are more resistant to rumen degradation (Varvikko, 1 986).

Interestingly, the amino acids which were least resistant to rumen degradation varied depending on the protein source; glutamic acid in rapeseed, methionine, alanine and glycine in barley, arginine and alanine in ryegrass and methionine, asparagine and tyrosine in barley straw were all degraded faster than the protein as a whole (Varvikko, 1986). However, the relationship between protein source and the rate of amino acid degradation in the rumen has not been resolved.

1.3.4.3 DISULPHIDE BONDS AND PROTEIN STABILITY.

Disulphide bonding occurs in a polypeptide chain between two cysteine residues. The reaction involves the free SH groups from cysteine only, the S group of methionine being protected by a CH3 group is unable to form a disulphide bond.

Matsumura et al. ( 1989) constructed mutants of phage T4 lysozyme, normally a disulphide free protein, which contained either 1,2 or 3 disulphide bonds. He reported that as the number of disulphide bonds increased, the melting temperature of the protein increased. As melting temperature is an index of protein stability, these results suggest that the presence of disulphide bonds increased protein stability. Treatment of mutant proteins to destroy disulphide bonds, reversed the trend. Nugent et al. ( 1983) reported that breaking the disulphide bonds in bovine serum albumin (BSA) with dithiothreitol, converted the protein

70 from being highly resistant to rumen degradation, to one which was readily degraded in the rumen. Therefore, the relatively high cysteine content of ovalbumin, PAl , and SF8 may be responsible for their resistance to rumen degradation.

1.3.4.4 RUMEN DEGRADATION MEASURED BY THE NYLON BAG TECHNIQUE.

Mehrez and Orskov ( 1977) proposed a method by which the degradability of dietary

protein could be measured. The method involved suspending nylon bags containing the test diet in the rumen for varying lengths of time. The rate of degradation of nitrogen or protein was equated to the rate of loss of protein or nitrogen from the nylon bag over time.

Estimates of the effective degradation of a protein can be calculated from an equation proposed by Orskov and McDonald (1979).

p = a + b (1 -e-ct)

where:

p = the effective degradation of the protein.

a = the instantly degradable fraction of the protein. b = the proportion of the protein degraded over time (t) at

a constant rate (c).

(a+b) = the potential degradability.

1.3.4.5 DEGRADATION VERSUS SOLUBILITY.

It has generally been assumed that proteins which disappear from a feed incubated in the rumen in a synthetic-fibre bag have been completely degraded (Ganev et aI., 1979).

Nugent and Mangan ( 1981) demonstrated that it was possible to study degradation of

individual proteins within a mixture of proteins during their incubation in the rumen, by SDS-polyacrylamide gel electrophoresis. This application is possible because the proteins of rumen microflora are extremely heterogeneous and do not interfere with detection of

added plant proteins (Spencer et a/., 1988). Using this approach, Nugent and Mangan,

(198 1 ), Nugent et al. ( 1 983) and Spencer et a/., ( 1988) demonstrated that the loss of protein or nitrogen from nylon bags suspended in the rumen was not necessarily correlated with the rate of protein degradation in the rumen. It would appear that the loss of nitrogen or protein from nylon bags suspended in the rumen is more correctly equated to the rate of

1 1. solubilisation of plant protein in rumen fluid, and that the rate of solubilisation is not necessarily a good measure of the rate of degradation of the individual protein components that constitute total plant protein.

1.3.5 GENETIC ENGINEERING.

1.3.5.1 GENETIC ENGINEERING : AN INTRODUCTION.

Amino acids cannot bind to DNA molecules, so a cell cannot produce a protein molecule directly from DNA, whilst there is less risk of damage to a cell's single DNA molecule if it is not directly used as a template for protein synthesis. Therefore, an intennediate molecule, messenger RNA (mRNA) is copied from DNA and then used repeatedly for protein

synthesis (Fig. 1 1).

1.3.5.1.1 The Genetic Code.

A DNA molecule consists of two deoxyribose-phosphate chains, which are coiled about one another to form a double-stranded helix. The chains follow the outer edge of the molecule and the nucleotide bases adenine (A), thymine (T), guanine (G) and cytosine (C) are in a helical array in the central core. The nucleotide bases from one strand are linked to nucleotide bases from the other strand by hydrogen-bonding to form the purine-to­

pyrimidine base pairs; A:T and G:c.

The sequence of amino acids in a polypeptide chain is ultimately coded for by the sequence of nucleotide bases in DNA. However, there are only four nucleotide bases but 20 amino acids. Therefore, triplets of nucleotides or codons specify each amino acid eg ... TGC specifies cysteine while AAG specifies lysine. The collection of codons that specify all the amino acids are known as the genetic code. The genetic code is nonoverlapping, therefore each codon specifies only one amino acid.

1.3.5.1.2 The Gene.

A segment of DNA molecule specifying a complete polypeptide chain is defined as a gene. Genes nonnally remain on chromosomes and do not directly serve as templates for protein biosynthesis, which takes place on the ribosomes. Instead, mRNA which contains the complementary codon sequence to the DNA, serves as the template, thus providing the genetic infonnation specifying the sequence of amino acids during protein biosynthesis. Figure 12 describes a chimeric gene, which differs from a natural gene in that it has been constructed in a laboratory using genetic engineering techniques. A chimeric gene, once