The Cysteine Biosynthetic Pathway

The Australian wool industry provides one useful area where this approach might succeed. When sheep are fed a diet that simulates that of the grazing animal, the amino acid cysteine is rate limiting for wool growth. Although cysteine itself is not an essential amino acid for mammals, it can only be synthesized from methionine. Thus, the supply of either methionine or cysteine is essential in the sheep diet. In order to increase the rate of wool growth, it is clearly necessary to increase the supply of one of these two amino acids. However, direct dietary supplementation of these amino acids is not effective as a method of increasing their supply, because the unique features of ruminant digestion ensure that added amino acid is degraded by the resident microflora. In the past, different approaches have been taken to overcome the degradation that occurs. For example, the ruminai degradability of proteins can be reduced by treatment with formaldehyde (23), which prevents ruminai degradation, but allows digestion in the distal part of the digestive tract. Alternatively, methionine itself can be encapsulated in a medium that survives the rumen, but not the lower digestive tract (23). However, these methods have not been economical in the practical farm environment.

An alternative approach is to introduce into the sheep genome the genes that encode the cysteine biosynthetic pathway. These genes are functional in prokaryotes and the auxotrophic eukaryotes, and are thus available for modification for expression in the digestive tract of sheep. The actual pathway for the biosynthesis of cysteine in E. coli is complex. It consists of two discrete parts, a pathway for the reduction of a sulfur source to sulfide, and a simple two-step pathway to combine the sulfide with the amino acid serine to produce cysteine. The genes that encode the second part of the process are cysE and cysK, encoding the enzymes serine transacetylase (SAT) and O-acetylserine sulfhydrylase (OAS). These catalyze the following reactions:

serine + acetyl-CoA -> O-acetylserine + CoA-SH serine transacetylase

O-acetylserine + H2S —> cysteine + acetate (1)

O-acetyl serine sulfhydrylase

The research involved in this type of genetic engineering project can be divided into four parts:

1. Isolation and characterization of the appropriate DNA coding and promoter sequences;

2. Construction and testing of various fusion genes in cell culture to determine the optimum configuration of the various components;

3 Testing of the preferred gene in transgenic mice to determine the efficiency and tissue-specificity of expression in vivo; and

4. Transfer of the preferred gene to transgenic sheep.

The cysE and cysK genes from E. coli have been isolated and fully characterized (24-26), thus providing the necessary coding sequences for the gene-transfer experiments. In order to modify the genes for expression in eukaryotic cells, the bacterial coding sequence has been inserted downstream from the sheep metallothionein-Ia promoter with the sheep growth-hormone gene used for stabilization of the mRNA transcribed from the fusion gene product (27,28). The general design of these genes is shown in Fig. 1. When mouse L-cells were transformed

Legend

1

MT Promoter

1

Bacterial gene

1

Growth Hormone

Fig. 1. Diagrammatic representation of the modifications made to bacterial coding sequences to provide expression in eukaryotic cells. Gene 1 contains excm 5 of the sheep growth-hormone gene 3' to the bacterial sequence. Gene 2 is similar, but contains the entire sheep growth-hormone gene. Gene 3 consists of a fusion of two gene 1 sequences, such that a single piece of DNA encodes the enzymes necessary for the cysteine synthesis or glyoxylate cycle biochemical pathways.

with these genes, mRNA transcripts of the predicted sizes were detected with probes encoding the appropriate cysE or cysK coding sequences (28). Extracts prepared from the transformed cells contained readily detectable levels of the enzymes SAT and/or OAS (Table 1).

The expression of the cyj-encoding fusion genes was examined in transgenic mice. Only those genes containing exon 5 of the sheep growth-hormone gene located 3' to the bacterial coding sequence were expressed at detectable levels in zinc-induced transgenic mice. This information is of general relevance to the expression of genes in transgenic animals, since the genes containing only exon 5 do not possess any exon/intron structure. Introns therefore are not an obligatory requirement for the expression of foreign genes in transgenic mice (but see 29).

Having established the fact that both genes can be independently transcribed and translated in transgenic mice, the transfer of both genes was examined. The transcription and translation of both E. co/i-derived sequences have been demonstrated in animals containing the combina-

Table 1

Activity of Serine Transacetylase (SAT) and O-Acetylserine Sulfhydrylase (OAS) in Extracts from Cells Transformed with Various Fusion Genes Containing Either the cysE (CE) or cysK (CK) Gene of Escherichia coli"

Zn-induced Uninduced

Construct SAT OAS SAT OAS

pMTCEKl 268 6960 86 1242

"Genes were constructed as shown in Fig 1 pMTCElO, pMTCK7, and pMTCEKl contain only exon 5 of the sheep growth-hormone gene pMTCEl 1 and pMTCKl 1 contain the complete sheep growth hormone gene Enzyme activity is expressed as |imoles substrate utilized (SAT) or product formed (OAS) /30 min /mg protein tion gene MTCEK1, which encodes both the cysE and cysK sequences in a single piece of DNA(Fig. 1). Zinc-inducible SAT and OAS activities (Table 2) were measured in the intestinal epithelium, the kidney, and the liver. Clearly, the actual biosynthesis of cysteine in these animals requires the presence of the necessary substrates in the tissues that express the transgenes, and current experiments are directed toward establishing whether such biosynthetic activity can be demonstrated in the mice. Since earlier studies have already indicated that genes that are expressed in transgenic mice are also expressed at high level in transgenic sheep, we have also commenced the transfer of the MTCEK1 gene to sheep.

Rogers et al. (24,30,31) have used a similar approach to achieve the same goal of enabling sheep to synthesize cysteine from H2S. In this case, the source of genes to provide the appropriate coding sequences is the bacterium, S. typhimurium. The two genes being used are the cysE gene, which is essentially the same as the E. coli gene, and the cysM gene, which encodes an OAS enzyme significantly different from that encoded by the cy.vATgene both in E. coli and S. typhimurium. After demonstrating expression of the genes in sheep cells in culture after being fused to the SV40 late promoter and SV40 polyadenylation signal sequences (30,31), they have been joined together in a single piece of DNA, and each coding sequence has been placed under the control of

Table 2

Activity of Serine Transacetylase (SAT) and 0-Acetyl serine Sulfhydrylase (OAS) in Tissue Extracts Prepared from Transgenic Mice"

Table 2

Activity of Serine Transacetylase (SAT) and 0-Acetyl serine Sulfhydrylase (OAS) in Tissue Extracts Prepared from Transgenic Mice"

Mouse line

Organ

SAT

OAS

CK7-26

Intestine

206

Kidney

352

Liver

13

CE 10-29

Intestine

6546

Kidney

0

Liver

0

EK1-28

Intestine

15144

519

Kidney

0

938

Liver

0

156

Brain

0

90

"CK7-26 contains the gene pMTCK7, CE10-29 contains pMTCElO, and EK1-28 contains pMTCEKl Specific activity is measured as nmoles substrate utilized (SAT) or product formed (OAS) / 30 min/mg protein

"CK7-26 contains the gene pMTCK7, CE10-29 contains pMTCElO, and EK1-28 contains pMTCEKl Specific activity is measured as nmoles substrate utilized (SAT) or product formed (OAS) / 30 min/mg protein a promoter derived from the long terminal repeat of the Rous sarcoma virus. This DNA has provided constitutive expression in transgenic mice and transgenic sheep, but unfortunately, not in tissues where the necessary substrate for the reactions might be expected. The research now in progress aims at providing the genes with a promoter more suited to directing their expression in tissues where the appropriate substrates for cysteine biosynthesis might be available (31).

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