Yvonne H. Edwards

A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine

Evidence suggests that intestinal apo-B48 is colinear with the amino-terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production, we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence, resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver, and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or posttranscriptional C----U change may result in the production of apo-B48, which represents the amino-terminal 2152 amino acids of apo-B100. This is the first example of tissue-specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.

Inherited variants of human nucleoside phosphorylase

1. A method, for the starch gel electrophoresis of human nucleoside phosphorylase (NP) is described. Multiple NP isozymes were found in most human tissues and the best resolution of these isozymes was achieved by electrophoresis in a buffer system containing lithium ions.

Susceptibility to spina bifida; an association study of five candidate genes.

Clues regarding candidate genes which influence susceptibility to spina bifida and anencephaly come from the identification of folate‐associated risk factors and from studies of mouse mutants showing neural tube anomalies. On this basis we selected five candidate genes; CBS, MS, MTHFR, T (Brachyury) and BRCA1 for genetic analysis in 31 Dutch and 48 British NTD families. Ten polymorphisms, two for each gene, were used in transmission tests for disequilibrium (TDT). In six instances more than 50 transmissions from heterozygous parents could be examined. Using TDT we find evidence for an association between an allele at the T gene and liability to NTD in the embryo. Data from British and Dutch populations showed the same trend and in combination gave a χ2TDT=4.89, P=0.03 (OR 2.39, CI 95% 1.02–5.61). No association, in either population group, was found for CBS, MS and MTHFR, the enzymes most directly associated with the known risk factors in folate metabolism. The possibility of complex genetic interactions was explored; the data show that a Gly919 MS variant occurs more frequently in combination with the MTHFR thermolabile variant in mothers of NTD offspring (OR 3.94, CI 95% 1.0–16.3).

Adenosine deaminase isozymes in human tissues

Several isozymes of adenosine deaminase (ADA) in human red cells can be detected by starch-gel electrophoresis and a number of inherited variants have been described (Spencer, Hopkinson & Harris, 1968). The phenotypes ADA 1, ADA 2-1 and ADA 2 occur in European populations with frequencies of about 0.9, 0.1 and 0.003 respectively, and are due to the two common alleles ADA1 and ADA2, phenotypes ADA 1 and ADA 2 representing the homozygotes and the ADA 2-1 phenotype the heterozygous genotype. Several less common phenotypes of red cell ADA have also been identified and in each case family studies have shown that the variant type represents the heterozygous combination of either ADA1 or ADA2 and a rare variant allele at the same locus (Hopkinson, Cook & Harris, 1969; Dissing & Knudsen, 1969; P. Gerald, 1969, personal communication; Detter et al. 1970). The present paper is concerned with the isozymes of ADA in human tissues other than red cells. The principal technique used was starch-gel electrophoresis and a wide range of tissues has been investigated. The sulphydryl group reactivities of the ADA isozymes in different tissues have also been compared, using the procedure of Hopkinson & Harris (1969)) and molecular size estimates of the various ADA isozymes have been obtained by gel filtration chromatography. I n most tissues isozymes similar to the red cell ADA isozymes were encountered but in many tissues additional isozymic forms were found which differed in electrophoretic behaviour, in sulphydryl group reactivity and also in molecular size from the red cell ADA isozymes. The results suggest that human adenosine deaminase may be determined by several different gene loci.

Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe.

A cDNA clone complementary to the mRNA encoding human myosin heavy chain has been isolated from a human fetal skeletal muscle cDNA library. A 600 base pair fragment of the inserted human cDNA has been used as probe in the Southern analysis of DNA from panels of rat/human and mouse/human somatic cell hybrids. All the sequences detected by this probe have been mapped to chromosome 17 in the region 17pter → 17p11. There was no evidence for MHC sequences on any other chromosome.

PGP9.5, A UBIQUITIN C-TERMINAL HYDROLASE ; PATTERN OF MRNA AND PROTEIN EXPRESSION DURING NEURAL DEVELOPMENT IN THE MOUSE

PGP9.5 is a neurone and neuro-endocrine specific ubiquitin carboxyl terminal hydrolase estimated to form 1-2% of total brain protein. We have examined the temporal and spatial distribution of PGP9.5 mRNA and protein in the developing mouse embryo. These studies show that PGP9.5 is present at high levels in all differentiated neurones throughout the central and peripheral nervous systems at all stages of development. The mRNA is detected in the neural tube 1 day prior to the protein and before neuronal differentiation is underway. Both mRNA and protein are present during the initial appearance of motor and sensory neurones, prior to their peak production. PGP9.5 immunoreactivity was detected using microwave pretreatment of sections in order to unmask epitopes. In general unmasking led to an overall enhancement of immunoreactivity although in some sites, for example the dorsal root and cranial nerve ganglia and the bundle of His, there was evidence for anatomical variation in the distribution of masked versus unmasked protein. The very early expression of PGP9.5 suggests that there is a role for ubiquitin hydrolases in the differentiation of neurone precursors as well as in the differentiated neurone.

Cloning and chromosome assignment of the human CDX2 gene

The caudal‐type homeobox gene Cdx2 encodes a transcription factor which is expressed in the intestine and is thought to play an important role in the proliferation and differentiation of intestinal epithelial cells. Mice heterozygous for null mutations in the caudal‐type homeobox gene Cdx2 show multiple adenomatous polyps in the proximal colon in addition to skeletal problems associated with abnormal segmentation. In human colorectal cancer the expression of both CDX2 and carbonic anhydrase 1, a gene regulated by CDX2, is reduced or absent. It is possible that mutation of CDX2is a primary event in the origin of some colorectal cancers. We have cloned human CDX2 cDNA and report here the nucleotide and protein sequences and assignment of the human gene to chromosome 13q12–13.

The detection and differentiation of the products of the human carbonic anhydrase loci, CAI and CAII using fluorogenic substrates.

1. The carbonic anhydrase isozymes of human red cells have been investigated by starch‐gel electrophoresis using the fluorogenic substrates, 4‐methyl‐umbelliferyl acetate and fluorescein diacetate, for the detection of the CAI and CAII locus isozymes respectively.

The distributions of subunit numbers and subunit sizes of enzymes: a study of the products of 100 human gene loci

1. A tabulation of subunit numbers and subunit sizes of a series of enzymes which have been studied electrophoretically in man is presented. The series of subunit numbers cover the isozyme products of 100 distinct gene loci. For 99 of these, estimates of subunit size are given. 2. The distribution of subunit numbers in the whole series is as follows: monomers, 28; dimers, 43; trimers, 4; tetramers, 24; octamers, 1. 3. The subunit sizes range from 13,000 to 116,000. The average subunit size for the whole series is close to 45,800. This corresponds to an average polypeptide chain length of about 425 amino acids. 4. No significant differences were found between the average subunit sizes for the separate classes of enzyme with different subunit numbers. 5. The enzymes were categorized in six different types according to the classification of the International Enzyme Commission. It was found that the oxido-reductases differed from the other types (transferases, hydrolases, lyases, isomerases, ligases) in showing a much greater proportion of multimeric enzymes. Only 1 out of 24 oxido-reductases appeared to be a monomer, whereas in the other enzyme types approximately one-third of the enzymes considered appeared to be monomers. No significant differences in subunit size were found between the various enzyme types. 6. It was found that, in general, where two or more separate gene loci are concerned in determining sets of isozymes with the same or very similar enzyme characteristics, there is a close correlation in subunit size and in most, though not all, cases the subunit numbers are the same.

Human enolase isozymes: electrophoretic and biochemical evidence for three loci

1 Four major enolase isozymes have been identified in human tissues and are referred to as L, M, ‘intermediate’ and ‘fast’. The M isozyme is the major form found in skeletal muscle and heart extracts and the L isozyme the major form found in extracts of liver and most other tissues The ‘intermediate’ and ‘ fast’ isozymes are most active in brain but are observed as weak components in most other tissues including heart but are not seen in skeletal muscle. It was observed that during fetal development of heart and skeletal muscle the L form declines in activity while the M form increases in activity. 2 The kinetic properties, heat stabilities and molecular sizes of the main enolase isozymes have been compared. Although the isozymes share many features in common, the ‘fast’ isozyme is more stable when subjected to heat treatment than either the L or M isozymes. Further, the ‘fast’ isozyme retains its dimeric structure and activity in the absence of magnesium ions while the L and M isozymes dissociate and lose activity. The ‘intermediate’ isozyme has properties which are intermediate to those of the L and ‘fast’ isozymes. 3 The ‘intermediate’ isozyme can be partially dissociated to equal quantities of L and ‘fast’ isozymes by storage at room temperature or by freezing and thawing in the presence of 2 m‐NaCl. Conversely, mixtures of L with ‘fast’ and M with ‘fast’ give rise to an ‘intermediate’ isozyme after freezing and thawing. 4 Evidence derived from this study has led to the suggestion that three separate gene loci are involved in the determination of human enolase. It is proposed that one of these, ENO1, determines the L isozyme which is the homodimer αα; another locus, ENO2, determines the ‘fast’ isozyme which is the homodimer ββ; and the third locus, ENO3, determines the M isozyme which is the homodimer γγ. The ‘intermediate’ isozyme seen as a strong component in brain and as a weak component in most other tissues is thought to be the heterodimer αβ. In heart however it is probably mainly βγ.