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R K Craig

Publications and source records attributed to R K Craig.

At least 55 records · Page 3Linked to original sources
PubMed1984

Guinea-pig casein A cDNA. Nucleotide sequence analysis and comparison of the deduced protein sequence with that of bovine alpha s2 casein.

The nucleotide sequence (1036 bases) of guinea-pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5' noncoding region, and the complete coding and 3' noncoding region. The remaining 5' noncoding sequence was obtained by primer extension. The deduced 223-amino-acid-coding sequence of guinea-pig pre-casein A exhibited 30% homology with bovine alpha s2 casein, the most striking similarities being in the locations of potential phosphorylation sites.

Amino Acid Sequence↗
PubMed1983

Katacalcin: a new plasma calcium-lowering hormone.

A second potent plasma calcium-lowering peptide, katacalcin (PDN-21), flanks calcitonin within the human calcitonin precursor. Plasma katacalcin was present in 57 healthy volunteers. Concentrations were higher in males than in females and approximately equimolar with calcitonin. Plasma katacalcin doubled within 5 min of calcium infusion. Plasma katacalcin was markedly raised in 20 patients with medullary carcinoma of the thyroid. Measurement of plasma katacalcin concentrations may prove useful in the diagnosis and follow-up of this condition. Katacalcin, like calcitonin, may be involved in both plasma calcium regulation and skeletal maintenance and thus may prove useful in the treatment of bone disease.

Adolescent↗
PubMed1983

Immunocytochemical localisation of katacalcin, a calcium-lowering hormone cleaved from the human calcitonin precursor.

Katacalcin is a newly discovered calcium-lowering hormone predicted from the nucleotide sequence of a cloned cDNA derived from human calcitonin mRNA. The aim of the study was to localise katacalcin by immunocytochemistry at both light and electron microscope levels. Antisera to synthetic katacalcin and calcitonin were used to investigate 8 cases of medullary thyroid carcinoma and 6 normal human thyroids (3 adult and 3 fetal). We have been able to demonstrate the co-localisation of these peptides in normal and neoplastic C-cells in all cases studied. Our results suggest that peptide sequences predicted by recombinant DNA technology can be localised using immunocytochemistry and that the combination of these techniques may have applications in diagnostic pathology.

Calcitonin↗
PubMed1982

Comparison of the nucleotide sequence of cloned human and guinea-pig pre-alpha-lactalbumin cDNA with that of chick pre-lysozyme cDNA suggests evolution from a common ancestral gene.

Nucleotide sequence analyses of essentially full-length copies of human and guinea-pig pre-alpha-lactalbumin cDNAs contained within recombinant plasmids, (i) confirm the presence of 19 amino acid hydrophobic amino terminal peptide extensions encoded within each mRNA; and (ii) provides evidence for the existence of a minor variant of guinea-pig alpha-lactalbumin mRNA encoding a protein with a 36 residue carboxyl-terminal extension. Comparison of the nucleotide sequence within the coding region of the human, and the predominant guinea-pig pre-alpha-lactalbumin mRNAs, with the analogous region of hen pre-lysozyme mRNA provides compelling evidence that all have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1981

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.

Calcitonin↗
PubMed1981

Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems.

1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.

Animals↗
PubMed1981

alpha-Lactalbumin is not a marker of human hormone-dependent breast cancer.

It has been proposed that milk protein gene expression in human breast carcinomas may indicate a functional oestrogen receptor mechanism, and may therefore be diagnostic of tumours responsive to endocrine therapy. Unfortunately this has not been proved, largely because of inconsistencies in the immunoassay procedures used to identify milk proteins, in particular alpha-lactalbumin, in tissue extracts or serum. Alternative procedures include the identification of milk protein mRNA by cell-free protein synthesis, and the identification of milk protein RNA transcripts by hybridization to a sequence-specific probe. Here we describe experiments using alpha-lactalbumin cDNA probes, purified using recombinant plasmids, which demonstrate that although polyadenylated and non-polyadenylated alpha-lactalbumin transcripts are present in normal human mammary tissue during pregnancy and lactation, alpha-lactalbumin transcripts are not detectable in the human tumour tissues studied. These experiments do however show that a peptide, which shares antigenic determinants with human alpha-lactalbumin is present in some breast tumour tissues.

Breast Neoplasms↗
PubMed1981

The construction, identification and partial characterization of plasmids containing guinea-pig milk protein complementary DNA sequences.

A complementary DNA (cDNA) plasmid library has been constructed in the plasmid pAT153, using poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland as the starting material. Double stranded cDNA was inserted into the EcoRI site of the plasmid using poly(dA . dT) tails, then transformed into Escherichia coli HB101. From the resulting colonies we have selected and partially characterized plasmids containing cDNA copies of the mRNAs for casein A, casein B, casein C and alpha-lactalbumin. However, the proportion containing casein C cDNA was exceptionally low, and these contained at best 60% of the mRNA sequence.

Animals↗
PubMed1981

Differential expression of alpha-lactalbumin and casein genes during the onset of lactation in the guinea-pig mammary gland.

1. The expression of alpha-lactalbumin and casein genes was examined in guinea-pig mammary tissue taken from animals both pre- and post-partum. 2. Analysis of total RNA by RNA excess hybridization with sequence-specific complementary DNA probes demonstrated that alpha-lactalbumin mRNA was present late in pregnancy, and that maximum concentrations were present at parturition. Casein gene transcripts were absent late in pregnancy (62 days), but by parturition were present at concentrations identical to those found at all time points examined throughout lactation. 3. Studies using mammary explants in organ culture showed that tissue from pregnant animals, or animals at parturition, synthesized and secreted only alpha-lactalbumin. After parturition, at the onset of casein synthesis, differential rates of secretion of alpha-lactalbumin and the caseins were observed. 4. The results are discussed in terms of the multiple intracellular mechanisms involved in the regulation of milk protein gene expression in the guinea-pig mammary gland.

Animals↗
PubMed1981

The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences.

We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153. Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA. Restriction enzyme maps were determined for six of these. Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin). Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.

Animals↗
PubMed1981

Characterisation of a membrane-bound serine-specific casein kinase isolated from lactating guinea-pig mammary gland.

Serine-specific and threonine-specific casein kinase activities have been identified in a Golgi-enriched membrane fraction isolated from the lactating guinea-pig mammary gland. The serine-specific casein kinase has been purified 2000-fold by affinity chromatography on ATP-agarose. The enzyme has an estimated Mr of 100000 as determined by sucrose gradient centrifugation and phosphorylates the serine residues of dephosphorylated guinea-pig caseins A and B in a qualitatively and quantitatively identical manner to caseins A and B secreted by lactating mammary gland explants in organ culture. The enzyme also phosphorylates casein C at serine, but not threonine residues. Studies on the relative location of the enzyme within a Golgi-enriched membrane fraction show that it is an integral component of the membrane, either in the form of a transmembrane protein or exposed on the luminal side of the membrane. Although casein kinase activity is not associated with the endoplasmic reticulum, it remains to be proven whether it is truly a Golgi enzyme, since analysis of subcellular membrane components fractionated by sucrose gradient centrifugation shows that the particulate protein kinase activity of the lactating mammary gland does not cosediment with galactosyl transferase, possibly a reflection of the heterogeneous nature of mammary gland Golgi apparatus. It seems likely that the serine-specific casein kinase activity described is responsible for the phosphorylation of caseins in the lactating guinea-pig mammary gland, and that this occurs after the sequestration of processed but unphosphorylated caseins within the lumen of the endoplasmic reticulum.

Animals↗
PubMed1980

Post-transcriptional regulation of gene expression in guinea pig tissues.

The formation of individual functional mRNA sequences in higher organisms requires many steps in addition to transcription. These include RNA splicing, polyadenylation, base modification, transport from nucleus to cytoplasm and assembly into polyribosomes. Various control mechanisms must also operate. These will function on a quantitative basis to account for the differing frequency of the various classes of cytoplasmic mRNAs, and also on a qualitative basis, because in higher organisms not all the nuclear poly(A)-containing RNA molecules are found in a cytoplasmic poly(A)-containing RNA population from the same tissue. During our studies on the mechanisms controlling the accumulation of the poly(A)-containing RNA sequences which occur with high and moderately high frequency in the cytoplasm of the lactating guinea pig mammary gland, it became apparent that > 75% of the 20,000 or so poly(A)-containing nuclear RNA sequences were not found in the cytoplasmic poly(A)-containing RNA fraction. Here we demonstrate that many of the poly(A)-containing RNA sequences retained in the nucleus of the lactating guinea pig mammary gland are also present in the nucleus and cytoplasm of the liver of the male guinea pig. These observations provide new evidence for a predominant role of post-transcriptional mechanisms in the regulation of structural gene expression in guinea pig tissue.

Animals↗
PubMed1980

Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland.

1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)-cellulose into three populations; those that bound at ;low salt' [long poly(A) tracts, 78-32 nucleotides]; those that bound at ;high salt' [shorter poly(A) tracts, 48-21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ;low salt' and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. (3)H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ;low-salt' post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ;low-salt' poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6x10(5) copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ;abundant' and so-called ;moderately abundant' sequences were present in essentially identical concentrations (2x10(3) copies of each sequence/cell). The majority of these (90-95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

Animals↗
PubMed1980

Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.

Animals↗
PubMed1980

Differential distribution of poly(A)-containing RNA sequences between the nucleus and post-nuclear supernatant of the lactating guinea-pig mammary gland.

RNA complexity analysis of poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland demontrates that the complexity within the nuclear population was three--four-fold greater than that in the equivalent post-nuclear polyribosomal population [see Craig et al., Biochem. J. (1979) 181, 737-756]. Up to 21 000 different sequences distributed in two abundance groups were detected in the nucleus, whereas 5000 sequences distributed in three abundance groups were present in the post-nuclear population. All poly(A)-containing RNA sequences present in the post-nuclear fraction could be detected in the nuclear poly(A)-containing population. Analysis of the relative distribution of the three post-nuclear abundance populations within the nuclear poly(A)-containing RNA population demonstrates that the abundant and moderately abundant post-nuclear sequences were present a similar concentrations within the nucleus, and comprised the abundant nuclear population. The abundant and moderately abundant post-nuclear sequences were present at 62200 and 785 copies of each sequence/cell in the post-nuclear fraction respectively, and 44 and 118 copies of each sequence/cell in the nuclear fraction respectively. The scarce post-nuclear sequences (18.5 copies of each sequence/cell) were also present at low levels in the nuclear fraction (0.1 copy of each sequence/cell). the results are discussed in terms of the post-transcriptional regulation of gene expression in the lactating guinea-pig mammary gland.

Animals↗
PubMed1979

Initiation and processing in vitro of the primary translation products of guinea-pig caseins.

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal 'signal'-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

Animals↗