Jim Haralambidis

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

The solid phase synthesis of oligonucleotides containing a 3′-peptide moiety

Abstract A hybrid comprising an oligodeoxyribonucleotide linked via the 3′-hydroxyl to the amino terminus of a peptide has been prepared using a combination of solid-phase FMOC peptide synthesis methodology and phosphoramidite chemistry.

Sites of expression and induction of glandular kallikrein gene expression in mice.

In order to provide a foundation for comparison across species of glandular kallikrein genes, we have studied the 12 functional mouse genes on the basis of expressing cell types, developmental patterns of expression and gene response to hormonal induction. We have shown expression of the renal kallikrein gene in the female anterior pituitary, the thick ascending limb of renal cortical distal tubules, nasal glands of neonatal mice and at varying levels throughout the duct tree of major salivary glands of immature and adult mice, except for intercalated ducts. This gene did not respond to hormonal induction in salivary glands. The other 11 of the 12 genes are expressed in androgen-responsive cells of granular convoluted tubules of the submandibular salivary gland from 22 days postnatal, when sexual dimorphism of expression first becomes apparent. Expression of these genes is induced prematurely in 22-day-old mice by treatment with testosterone or thyroxine. In the adult female mouse, estrogens also induce elevated levels of expression. One of the glandular kallikrein genes is expressed in Leydig cells of the testis as well as the submandibular gland. This study has extended the basis for cross-species comparison of glandular kallikrein genes.

Location of glandular kallikrein mRNAs in mouse submandibular gland at the cellular and ultrastructural level by hybridization histochemistry using 32P- and 3H-labeled oligodeoxyribonucleotide probes.

We investigated the location of expression of mouse glandular kallikrein genes in the submandibular gland of adult male mice at the ultrastructural level by hybridization histochemistry, using 32P- and 3H-labeled oligodeoxyribonucleotide probes. Vibratome slices were hybridized, flat-embedded, sectioned, and autoradiographs prepared. The 32P-labeled probe, which was specific for a region common to all mouse glandular kallikrein mRNAs, provided resolution at the cellular and subcellular level, demonstrating mRNA transcripts encoded by the majority of the 12 mouse glandular kallikrein genes in the perinuclear area of granular convoluted tubule cells (GCT) associated with rough endoplasmic reticulum (RER). The 3H-labeled probe was specific for mRNA transcripts of mGK-6, the renal kallikrein gene, which is also expressed in salivary glands. Occasional morphologically distinct granulated cells within GCTs, as well as striated duct cells, were found to express this gene. Resolution obtained with this 3H-labeled probe showed mGK-6 mRNA in striated duct cells to be located on RER and in the nucleus and perinuclear area of the cell. There was also an apparent mitochondrial association with regions of RER that labeled with this probe. The location of hybrids was confirmed by simultaneous assessment of sites of silver grains in serial sections. There are therefore at least two types of mGK-6-expressing cells in ducts of the submandibular gland, which are distinct from those expressing other kallikrein genes. In striated duct cells, there is evidence of a close mitochondrial association with RER that contains labeled mGK-6 transcripts, which is unexplained.

The Design and Synthesis of N 4-Anthraniloyl-2′-dC, the Improved Syntheses of N 4-Carbamoyl-and N4- Ureidocarbamoyl-2′-dC, Incorporation into Oligonucleotides and Triplex Formation Testing

Abstract Three modified nucleosides were designed with the aim of achieving triplet formation with the CG base pair of duplex DNA. Direct anthraniloylation of 2′-deoxycytidine, using isatoic anhydride, afforded the novel N 4-anthraniloyl-2′-deoxycytidine. Much improved preparations of N 4-carbamoyl-2′-deoxycytidine and of N 4-ureidocarbonyl-2′-deoxycytidine were accomplished. The modified nucleosides were incorporated into oligonucleotides. Thermal denaturation studies and gel mobility shift analysis suggest that these nucleosides do not form base triplets with any of the four base pairs of DNA.

[14] – Location of Gene Expression in Tissue Sections by Hybridization Histochemistry Using Oligodeoxyribonucleotide Probes

Publisher Summary This chapter explores synthesis, labeling, and use of oligodeoxynucleotides in the technique of hybridization histochemistry. The technique of hybridization histochemistry is simple and convenient and has been used for several years for a variety of research topics and myriads of specimens with consistent success. Oligodeoxyribonucleotides are routinely synthesized by the solid-phase method on an Applied Biosystems Inc. 380A DNA synthesizer, using phosphoramidite chemistry. The most convenient method for the routine purification of large numbers of oligonucleotides is polyacrylamide gel electrophoresis (PAGE). The choice of radioisotope used for incorporation into the oligonucleotide depends on the experimental requirements. The temperature for hybridization of sections with oligodeoxynucleotides is related to the probe length, specificity of the probe for the target mRNA, and the formamide and salt concentration of the hybridization buffer. The technique of hybridization histochemistry may be used in conjunction with immunohistochemistry for the investigation of transcriptional regulation of neuroendocrine peptides and identification of the peptide product.

The preparation of enzyme-labelled oligonucleotides by reductive amination

Abstract Oligonucleotides containing an aldehyde group at the 3′-terminus have been prepared using a derivatized solid phased. This solid phase contained a short peptide that included a lysine residue substituted at the N e -amino group with carboxybenzaldehyde. The oligonucleotide was coupled to alkaline phosphatase under reductive amination conditions to produce a conjugate with unaltered hybridization properties.

HYBRIDIZATION HISTOCHEMISTRY - LOCATING GENE EXPRESSION

A technique we have called hybridization histochemistry has been developed for the location of specific mRNA populations in specially prepared sections of tissue (Hudson et al 1980; Coghlan et al 1981; Coghlan et al 1984; Hudson et al 1981; Jacobs et al 1983; Coghlan et al 1984). Later the same approach has been used by others to identify specific neurones in the hypothalamus (Gee et al 1983), to study the origin and fate of identified neurones in aphysia (McAllister et al 1983), location of specific genes in Drosophila embryos (McGinnis et al 1984), and enkephalin in the adrenal (Block et al 1984).

Oligonucleotide-Polyamide Conjugate Probes with Multiple Non-Radioactive Labels: Applications and Comparison to Radiolabelled Probes

Abstract Oligonucleotide-polyamide conjugate molecules containing multiple biotin labelled polyamide have been used as non-radioactive probes. We found that these probes have similar sensitivity to 32p labelled probes. Chemiluminescent detection is the method of choice.

POLYAMIDE-OLIGONUCLEOTIDE HYBRID MOLECULES

1. Recent experience with polyamide–oligonucleotide hybrids shows the versility of this approach for replacement of radioactive isotopes for detection of hybridization on gels and for hybridization histochemistry.