Norman Hardman

Molecular cloning of a cDNA for human pregnancy-specific β1-glycoprotein: homology with human carcinoembryonic antigen and related proteins

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.

Characterization of foldback sequences in hamster DNA using electron microsocpy.

Foldback sequences in nuclear DNA from cultured Hamster fibroblasts (BHK-21/C13 cells) have been characterized by electron microscopy. One half of the structures observed when denatured hamster DNA is allowed to anneal in the range O less than Cot1 less than 1 x 10(-4) M sec result from the annealing of inverted sequences forming foldback DNA. The remainder have a probable bimolecular origin. arising from rapidly-annealing sequences of satellite-like complexity. The average length of the inverted sequences in the foldback molecules is about 0.9 kilobases. There is estimated to be about 42,000 such sequences (21,000 pairs) in the hamster genome, approximately 45% of which form looped structures with a mean loop length of 1.74 kilobases. Contrary to previous reports, binding of the renatured duplex molecules to hydroxyapatite results in a poor recovery of structures containing identifiable foldback sequences, due to preferential enrichment of the bound fraction with duplexes formed by intermolecular annealing.

Characterisation of ribosomal satellite in total nuclear DNA from Physarum polycephalum

The distinctive properties of satellite DNA molecules containing the genes for ribosomal RNA in Physarum polycephalum permits their identification in total, unfractionated nuclear DNA in the foldback form, after denaturation and fast annealing. Using the electron microscope the location and properties of three characteristic regions containing tandemly-repeated, inverted sequences have been investigated. At least two additional regions, also containing tandem repeats, are shown to be present and located towards each end of the rDNA molecule, at a site adjacent to the segment coding for the 26 S rRNA. All the regions which contain tandem repeats are composed of sequences which, within experimental error, appear to share a common unit repeat length of about 90 nucleotides.

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

A family of long, highly‐repetitive sequences, referred to previously as ‘HpaII‐repeats’, dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one‐half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20‐50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the ‘HpaII‐repeat’ forms part of a larger repetitive DNA structure, ∼8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition‐like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small ‘cruciforms’ or ‘Z‐DNA’ structures which might be recognised during the recombination process.

Structural organization of a hypermethylated nuclear DNA component in Physarum polycephalum.

Digestion of Physarum polycephalum nuclear DNA using the restriction endonuclease HpaII generates two components, distinguishable on the basis of their molecular size. The high-molecular-weight, HpaII-resistant component, which accounts for 20% of the DNA, contains a fivefold greater concentration of 5-methylcytosine residues than the low-molecular-weight HpaII-digested fraction. Segments of hypermethylated (M+) DNA are largely composed of a single, long, highly repeated sequence, and this major element is sometimes associated with other less highly repetitive sequences in the M+ DNA fraction. Restriction mapping of cloned Physarum M+ DNA segments, and Southern blot analysis of genomic DNA using subcloned segments of M+ DNA as a probe, provide evidence for sequence variation within different copies of the dominant highly repeated element, and possibly the other associated repeats in M+ DNA, and additionally that almost complete tandemly repeated copies of the major repeat are found in some M+ DNA segments.

Isolation and characterisation of a variant allele of the gene for human Apolipoprotein E

Size-selected human DNA fragments enriched in the Apolipoprotein E (ApoE) gene sequence were cloned from an individual of known ApoE phenotype, E2/E2. The clone bank was screened using a human cDNA clone for the ApoE locus (1), and a single genomic clone was isolated. Sequence data obtained from appropriate subcloned fragments confirmed that the codon for Arg-158 (CGC) in the E3 allele is altered to the codon for Cys (TGC) in the E2 allele. Hybridisation data indicated the presence of at least one intron in the ApoE gene, consistent with the structure of an independently isolated human ApoE4 allele (2).

Molecular Organization of the Physarum Genome

Physarum polycephalum, because of its unique life cycle, is a useful organism in which to address a wide variety of questions of biological interest. These include problems relating to motility, differentiation, cell cycle regulation, and ultrastructural organization of the mitotic apparatus. As reviewed in other chapters of this volume, much progress has already been made on several fronts in these and other areas. In many instances, as stressed repeatedly in this Workshop, further progress will depend on the development of procedures that provide the experimental tools necessary for rigorous molecular genetic analysis in Physarum. In other organisms, such procedures have depended for their success on the identification of important regulatory sequences and novel genetic elements, providing a means to develop appropriate vectors for the cloning, in vitro manipulation, reintroduction, and controlled expression of important genes. This information can be obtained only from studies of the structure and organization of genomic DNA sequences. Thus, apart from its intrinsic interest, the molecular characterization of the Physarum genome will have important implications for future studies of the many facets of Physarum biology.

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences.

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hybridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.

The Effect of Age on the Properties of Poly(A)-containing Messenger RNA in Physarum polycephalum

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.