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Biomedical subjects

A L Archibald

Publications and source records attributed to A L Archibald.

At least 73 records · Page 4Linked to original sources
PubMed1991

Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice.

We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.

Animals↗
PubMed1991

Cosegregation of porcine malignant hyperthermia and a probable causal mutation in the skeletal muscle ryanodine receptor gene in backcross families.

A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor (RYR1) gene that was previously shown to be correlated with MG in five Canadian swine breeds. Cosegregation of the mutation with MH in 338 informative meioses led to a lod score of 101.75 for linkage at Omax = 0.0. The substitution was also associated with a HinPI- BanII+ RsaI- haplotype in this breed, as in the five breeds tested earlier, suggesting its origin in a common founder animal. DNA-based detection of the MH status in 376 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation.

Animals↗
PubMed1990

High-level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice.

Reduced circulating levels of alpha 1-antitrypsin (alpha 1 AT) are associated with certain alpha 1 AT genotypes and increased susceptibility to emphysema. Unfortunately, the amounts of alpha 1 AT that would be required for replacement therapy are beyond the capacity of plasma fractionation and mammalian cell culture systems. Thus, we have examined the potential of transgenic animals as an alternative means of producing human alpha 1 AT. A hybrid gene constructed by using sequences from the ovine milk protein gene beta-lactoglobulin fused to an alpha 1 AT "minigene" was used to generate transgenic mice. Of 13 independent transgenic mice and mouse lines, 5 expressed the hybrid gene in the mammary gland, 5 in the salivary glands, and 2 in both these tissues. Human alpha 1 AT was secreted into the milk of each of the 7 mice and mouse lines that expressed the hybrid gene in the mammary gland. Four of these mammary-expressing transgenic mice and mouse lines produced concentrations of at least 0.5 mg of alpha 1 AT per ml in their milk; one line (AATB 35) produced 7 mg of this protein per ml. alpha 1 AT from transgenic mouse milk was similar in size to human plasma-derived alpha 1 AT and showed a similar capacity to inhibit trypsin. Expression at equivalent levels in transgenic sheep or cattle would yield sufficient alpha 1 AT for therapeutic purposes.

Animals↗
PubMed1990

Localization of the PGD and TGF beta-1 loci to pig chromosome 6q.

The TGF beta-1 and PGD loci have been localized by in situ hybridization to the C-greater than q2.1 and q2.2 -greater than q2.5 regions of pig chromosome 6. These assignments confirm that the conversation of syntenic groups around GPI and PGD extends to pigs where these two groups are uniquely found to be linked. Our data also support the hypothesis that the porcine and human inherited malignant hyperthermia syndromes are caused by mutations in homologous genes which map to human chromosome 19q, porcine chromosome 6q and murine chromosome 7.

Animals↗
PubMed1990

Modification of milk composition.

Revolutionary new opportunities for the modification of milk composition have been created by the development of methods for gene transfer and targeted mutation of genes may extend the range of opportunities still further. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue-specific hormonally induced expression in the mammary gland. Fragments of the ovine beta-lactoglobulin gene fused to cDNA for the human therapeutic proteins clotting factor IX and alpha-1 antitrypsin have directed production of these proteins in the milk of transgenic mice and sheep. Factor IX was biologically active and co-migrated with authentic proteins, but was present at too low a concentration for commercial exploitation. Recent observations suggest that fusion genes containing genomic clones direct production of higher concentrations of protein. Mouse whey acidic protein genomic sequences also directed production of low concentrations of human tissue plasminogen activator in the milk of transgenic mice. Targeted expression of this kind may be used for the production of therapeutic and industrial proteins, to increase the concentration or modify the nature of milk proteins, reduce the concentration of lactose, change the composition of fat or direct production of bacteriocidal proteins in milk in order to combat mastitis.

Animals↗
PubMed1989

The molecular manipulation of milk composition.

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.

Animal Husbandry↗
PubMed1988

A new allele at the Pgd locus in pigs.

A new electrophoretic variant of porcine 6-phosphogluconate dehydrogenase (PGD) is described. The new variant, PGD C, has been shown to be controlled by a third allele, PgdC, at the Pgd locus.

Alleles↗
PubMed1987

Comparison of the serum amylases of farm animals.

1. Serum isoamylases with alpha-glucosidase activity from cattle, sheep, horses, goats, red deer, pigs and dogs were compared to one another. 2. The isoamylases from cattle and pigs were polymorphic. 3. In agarose gel electrophoresis the isoamylases behaved as alpha-1-globulins but in starch gel electrophoresis they were differentially retarded by affinity effects. 4. Molecular weights were estimated: cattle (417,000); sheep (402,000); horses (420,000); goat (399,000); red deer (405,000); pigs (375,000) and dogs (390,000). 5. Isoelectric points were estimated: cattle, sheep, goat and red deer (pH 3.5); pig (pH 3.6); dog (pH 3.7) and horse (pH 3.9).

Animals↗
PubMed1986

A single nucleotide difference at the 3' end of an intron causes differential splicing of two histocompatibility genes.

The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed.

Amino Acid Sequence↗
PubMed1986

A new transferrin allele in sheep.

An electrophoretic variant of sheep transferrin, TfL, has been described. Transferrin L has been shown to be controlled by a single codominant allele, TfL, at the Tf locus. Transferrin L is electrophoretically distinguishable from the very similar transferrin TfKCzech. The value of gradient polyacrylamide gel electrophoresis for transferrin phenotyping in sheep is discussed.

Alleles↗
PubMed1984

Complete nucleotide sequence of the murine H-2Kk gene. Comparison of three H-2K locus alleles.

We have determined the DNA sequence of the H-2Kk gene of the mouse major histocompatibility complex (MHC). Comparison on the nucleotide and protein level of three H-2K alleles (Kk, Kb and Kd) reveals a high degree of homology, in particular between the Kb and Kk alleles. Differences between the two latter antigens are almost exclusively confined to the alpha 1 and alpha 2 domains. At nine positions in the extracellular part of the molecules we have found allele-specific amino acids. Interestingly, 78% of these residues are either polar or carry hydroxyl-groups. This makes it likely that they are exposed on the surface of the molecules and might then be part of antigenic determinants. We have also identified potentially allele-specific nucleotide sequences of the K genes which might be used as specific DNA probes.

Alleles↗
PubMed1983

Testing genetic models in populations which contain pedigree errors.

In a genetic analysis of a polymorphic system, differences between the observed type of an individual and that expected from the parental types can arise either from an incorrect model or from pedigree errors. Such pedigree errors can cause severe difficulties in studies of the mode of inheritance of a novel polymorphic system. A method is proposed which overcomes the problem by including sire and dam error rates explicitly in the genetic model. The error rates are estimated by maximum likelihood, and likelihood ratio tests used to compare different models or estimates from different data sets. The proposals are applied to a study of the inheritance of the bovine serum AmI amylases.

Animals↗
PubMed1982

Further studies on bovine serum amylase. 3. Affinity chromatography.

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.

Amylases↗
PubMed1981

Further studies on bovine serum amylase. 2. Affinity electrophoresis.

The polymorphism of bovine serum amylase, which is controlled by the AmI locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the AmI isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different AmI phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the AmI polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the AmI isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the AmI isozymes is, therefore, another example of affinity electrophoresis. All the AmI amylases have been shown to share a common isoelectric point of pH 3.5.

Amylases↗
PubMed1980

Is inosine the physiological energy source of pig erythrocytes?

Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.

Adenosine Deaminase↗