Raymond J. MacDonald

Number and evolutionary conservation of α- and β-tubulin and cytoplasmic β- and γ-actin genes using specific cloned cDNA probes

Abstract Bacterial clones containing inserted DNA sequences specific for α-tubulin, β-tubulin, β-actin and γ-actin have been constructed from mRNA of embryonic chick brain. Plasmids containing approximately 75, 90 and >90%, respectively, of the sequences present in α-tubulin, β-tubulin and β-actin mRNAs have been isolated as well as clones containing parts of the extensive 3′ untranslated regions of the β- and γ-actin mRNAs. The sequences for the two tubulins do not cross hybridize. Hybridization of labeled, cloned probes for each of the tubulins with chicken DNA digested with several restriction endonucleases reveals about four fragments for α- and four for β-tubulin. This seems to be the number of genes, since both the 5′ and 3′ ends of either cloned tubulin cDNAs hybridize to at least four common fragments in genomic DNA which has been digested with restriction endonucleases. The tubulin probes are able to hybridize under stringent conditions to DNA of all vertebrate genomes tested, as well as to sea urchin DNA, but not to yeast DNA. In digested sea urchin sperm DNA there are more than 20 different fragments which hybridize to both the 5′ and 3′ ends of the tubulin cDNAs. A full-length β-actin cDNA clone hybridizes to 4–7 bands in restricted chicken DNA and cross hybridizes to DNA from every other species tested, including sea urchin and yeast. Hybridization to chicken DNA of cloned probes specific for the 3′ untranslated regions of β- and γ-actin mRNA indicates that the β sequence is present only once in the genome and the γ is present in at most three copies. Neither 3′ untranslated sequence is conserved evolutionarily.

[5] Cloning of hormone genes from a mixture of cDNA molecules☆

Publisher Summary This chapter discusses the cloning of hormone genes from a mixture of complementary deoxyribonucleic acid (cDNA) molecules. The molecular cloning of cDNA synthesized from a purified messenger ribonucleic acid (mRNA) is a well-established method for obtaining purified DNA for sequence analysis and use as a hybridization probe. Only a limited number of purified mRNAs are currently available that include those from specialized cells producing predominantly a single protein, such as globin, immunoglubin, or ovalbumin. The chapter describes the procedures used to isolate and clone specific hormone cDNAs from an impure mRNA population. The method employs the restriction endonuclease cleavage of double-stranded cDNA transcribed from a complex mixture of mRNA. The method does not require any extensive purification of RNA but instead makes use of the transcription of RNA into cDNA, the sequence-specific fragmentation of this cDNA, with one or two restriction endonucleases, and the fractionation of the cDNA restriction fragments on the basis of their lengths. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneous-length DNA fragments from any cDNA species that contains at least two restriction sites. From the initially heterogeneous population of cDNA transcripts, uniform-sized fragments of desired sequence are produced. The chapter discusses a complete scheme for cDNA cloning.

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins.

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.

Comparative analysis of zymogen granule membrane polypeptides

Abstract Polypeptides of zymogen granule membranes from rat, beef, dog, pig, and rabbit pancreas were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. Granule membranes were characterized by a single major polypeptide species detected by the periodic acid-Schiff procedure. The relative mobilities of this component were similar for these mammals. The component was distinct from major polypeptides of mitochondrial or microsomal membranes. The zymogen granule membrane may, therefore, represent a highly specialized intracellular membrane.

Purification and partial characterization of an integral membrane glycoprotein from zymogen granules of dog pancreas

Glycoproteins, long known to be functional constituents of cell surfaces, have recently been detected in intracellular membranes [ 1 ] . Membranes from the rough endoplasmic reticulum and the Golgi complex [2,3] and membranes from storage granules [4-61 of various secretory tissues, possess glycoproteins. Several functions for such intracellular, membrane-bound glycoproteins can be proposed. They may be organelle-specific and may function as determinants in membrane-membrane interactions, such as fusion. Alternatively, or additionally, membrane constituents may flow from the rough endoplasmic reticulum to the smooth endoplasmic reticulum and then to membrane-bound vesicles. Fusion of the vesicle membrane with the plasma membrane would provide a mechanism for the insertion of plasma membrane components [7,8]. According to this latter view, storage-granule membranes would contain precursors to plasma-membrane glycoproteins. The lack of detailed information regarding specific intracellular membrane glycoproteins has impeded exploration of these possibilities. Pancreatic zymogen granules are specialized organelles for the temporary storage of secretory proteins. Although zymogen granules arise from the Golgi complex and fuse with the plasma membrane during secretion, the molecular mechanisms underlying these

Phosphorylation of a zymogen granule membrane polypeptide from rat pancreas

Secretion of digestive enzymes and proenzymes by the exocrine pancreas is stimulated by the polypeptide hormone, pancreozymin, and by acetylcholine [ 1,2] . Adenosine 3’:5’ cyclic monophosphate (cyclic AMP), butyryl derivatives of cyclic AMP and theophylline stimulated pancreatic secretion in vitro, suggesting that cyclic AMP is an intracellular mediator of these hormones [3]. The role of cyclic AMP in the pancreas is still not clear [4,5] . A possible function is the regulation of protein kinases. A soluble, cyclic-AMP dependent protein kinase was partially purified from beef pancreas [6] . Furthermore I-ambert et al. [7] observed that peptide hormonestimulated secretion in pancreatic tissue slices was coupled to increased phosphorylation of zymogen granules. We now report that purified zymogen granule membranes from rat pancreas phosphorylated a granule membrane polypeptide, distinct from the major polypeptide of this membrane [8,9]. This phosphorylation, apparently due to an endogenous protein kinase, was not stimulated by cyclic AMP. There was minimal stimulation by guanosine 3’: 5’ cyclic monophosphate (cyclic GMP).

Pancreas‐specific genes: Structure and expression

Via recombinant DNA technology the mRNA sequence of pancreatic amylase has been cloned and its nucleotide sequence has been determined. The cloned sequence represents 96% of the total length of amylase mRNA; missing are an estimated 75 ± 30 nucleotides from the 5′ end. The amino acid sequence of rat pancreatic amylase was deduced solely from the nucleotide sequence of the mRNA. Unlike other eukaryotic mRNAs, the amylase mRNA has short 5′ and 3′ untranslated regions, suggesting that long untranslated regions of eukaryotic mRNAs either do not contain extensive functional sequences or that these sequences are incorporated within the amino acid coding region of amylase mRNA. The cloned amylase mRNA sequence was radiolabeled and used as a probe for in situ hybridization. These experiments demonstrate that amylase mRNA is present in all acinar cells but not in other pancreatic cell types. Using the cloned amylase mRNA sequences as a hybridization probe, three nonoverlapping genomic DNA fragments containing amylase gene sequences were isolated. From the similar sequence organization of the three amylase genes visualized by DNA heteroduplex mapping, a consensus structure of a rat amylase gene is proposed. It is an extended gene structure 10 kilobase pairs in length containing the 1547 base pairs of the cloned mRNA coding sequence interrupted by seven intervening sequences ranging from 400–2000 base pairs long. Thus, in nuclear DNA the amylase mRNA coding sequence is disrupted into at least eight segments from 150–300 base pairs long.