Design considerations for array CGH to oligonucleotide arrays.

BACKGROUND: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. METHODS: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. RESULTS: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. CONCLUSION: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

Isolation of genomic fragments from polymorphic regions by representational difference analysis.

Representational difference analysis is an effective technique for isolating the differences between two nearly identical genomes. We have found the technique to be extremely valuable in our analyses of mouse germline mutations. We also present several technical improvements in the procedure that make it more efficient and reliable.

Recovery of probes linked to the jcpk locus on mouse chromosome 10 by the use of an improved representational difference analysis technique.

Representational difference analysis (RDA) is a subtractive hybridization technique by which the differences between two complex genomes can be isolated. An improved version of this technique was used to isolate DNA segments that map to a narrow genetic region adjacent to the jcpk locus on Chromosome 10 of the mouse. A mutation at this locus acts recessively and causes an early onset polycystic kidney disease. Genomic subtractions involving DNA from C57BL/6 (B6) and its partially congenic partner, B6-jcpk/jcpk, produced 39 restriction fragments (difference products), 25 of which were unique and represented differences in BglII sites between these two strains. Although none identified the jcpk locus itself, 7 of these were mapped to an interval between 3.4 and 6.5 cM distal to the jcpk locus. Five of these 7 difference products were developed by subtracting B6-jcpk/jcpk from B6 DNA, but only 1 of the 5 was isolated using the original RDA technique. The other 4 were obtained by an improved technique that included size selection of difference products after the third round of subtractive hybridization and amplification. The remaining 2 of the mapped products resulted from the reciprocal subtraction experiment using the improvements. Thus, by this improved technique and two-way subtraction, we were able to add seven new markers to a relatively small genetic region on Chromosome 10.

Comparison of the ability of normal mouse mammary tissues and mammary adenocarcinoma to cleave rat prolactin.

Previous studies have shown that target tissues cleave rat prolactin (rPRL) to form a two-chain derivative that yields approximately 16- and approximately 6-kDa fragments upon reduction. Both cleaved rPRL and the purified 16-kDa fragment have novel biological activities. Thus, cleavage may be of physiological significance. We determined whether normal mammary tissue or stroma (lacking mammary epithelial cells [MEC]) or mammary tumor tissue could cleave rPRL in vitro. Tumor explants were derived from a transplantable mammary tumor cell line (TPDTMT-4EP). The hormone was incubated with explants of those tissues from female BALB/c or DDD mice. Medium samples were processed by SDS-PAGE, and the relative abundance of intact and cleaved rPRL was determined by densitometric analysis of immunoblots using an antiserum to the 16-kDa fragment of rPRL. After 2-hr of incubation, normal DDD mammary explants cleaved approximately 25% of added rPRL, but tumor explants cleaved none. Explants of intact virgin BALB/c mouse mammary gland and of parenchyma-free "cleared" mammary fat pad cleaved rPRL equally well, but explants of abdominal adipose tissue had low cleaving activity. Homogenates of cleared mammary fat pads that were incubated with rPRL contained a high proportion of the cleaved form but none of the free 16-kDa fragment. These results indicate that cleavage of rPRL is highly specific to mammary stroma. Such cleavage may yield a form of the hormone that has novel activities within the mammary gland or elsewhere in the body.

Cleavage of rat prolactin occurs within rat mammary tissue, and the cleaved form is present in serum but not in milk.

The present study was undertaken to determine whether cleavage of rat prolactin (rPRL) occurs within rat mammary tissue and to investigate whether the cleaved form and its 16-kDa amino terminal fragment are present in rat serum and milk. The relative abundance of the different forms of rPRL was determined by SDS-PAGE and immunoblot analysis using an antiserum to the 16-kDa fragment. Explants of mammary glands from lactating rats were incubated with intact rPRL. The tissue was then washed extensively to remove external PRL forms. The ratios of cleaved to intact rPRL in mammary tissue homogenate, in mammary-conditioned medium, and in serum from lactating rats were 4.2, 0.44, and 0.16, respectively. Although intact rPRL was found in milk, the cleaved form was not detectable. The 16-kDa fragment of rPRL was not detected in concentrated extracts of either serum or milk. These results indicate that lactating rat mammary tissue can internalize and cleave rPRL, but the cleaved form does not appear in milk. The cleaved rPRL is present in rat serum, but no evidence was obtained for the presence of the 16-kDa fragment of rPRL in either serum or milk.

Mass spectrometric analysis of the fragments produced by cleavage and reduction of rat prolactin: evidence that the cleaving enzyme is cathepsin D.

The site(s) at which mammary tissue enzymatically cleaves rat (r) PRL, and the possibility that the cleaving activity is cathepsin D, were investigated using mass spectrometry and enzyme inhibitors. Cleavage of intact rPRL [22,566 atomic mass units (amu)] by either mammary gland-conditioned medium or cathepsin D (both at pH 3) reduced the mass of the molecule by 397 amu. Subsequent reduction of the large rPRL fragment cleaved by either method generated two fragments of 16,364 and 5808 amu. The mass of the smaller fragment is consistent with the report of Vick et al. (Biochim Biophys Acta 931: 196-204, 1987) that its amino-terminal residue is Ser149. These results indicate that both enzyme preparations cleave rPRL by excision of the tripeptide Leu-Val-Trp (mass = 397 amu) between residues 145 and 149. The ability of both enzyme preparations to cleave rPRL at pH3 was inhibited by pepstatin A but not by phenylmethane sulfonyl fluoride, and both preparations were essentially inactive at pH7. Accordingly, the PRL-cleaving activity of rat mammary tissue is probably cathepsin D.

Processing of rat prolactin by rat tissue explants and serum in vitro.

Previous work has shown that enzymes from rat liver or mammary gland can cleave rat (r) PRL to form a two-chain derivative that yields approximately 16- and 7-kilodalton (kDa) fragments upon reduction. Both cleaved rPRL and the purified 16-kDa fragment have maintained biological activity. Thus, cleavage may be of physiological significance. To determine whether rPRL can be cleaved by intact cells, and to evaluate the extent to which rPRL processing is tissue specific and varies with physiological state, rPRL was incubated with slices of different tissues from cycling, midpregnant, or 15-day lactating rats. The molecular mass and relative abundance of rPRL and fragments of the hormone in the medium were determined using reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with an antiserum to the 16-kDa fragment of rPRL. Parenthetically, the fragment that was previously identified as having a molecular mass of 16 kDa had the same mobility on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the form that we designate as having a molecular mass of 14 kDa. Kidney, spleen, mammary gland and liver explants from pregnant rats processed PRL in qualitatively and quantitatively different ways. Mammary gland from lactating rats produced more of a 14-kDa fragment than liver or kidney slices from lactating rats, and the capacity to produce 14-kDa PRL by mammary gland from rats in different physiological states was as follows: pregnant greater than cycling greater than lactating. Fragments of 11 and 16 kDa were also produced, but only by mammary gland from lactating rats, and the latter only after 3 h of incubation. After a 2-h incubation, PRL-like immunoreactivity in lactating mammary gland medium was composed of 23-, 14-, and 11-kDa forms in the following relative amounts: 65 +/- 9, 17 +/- 1, and 10 +/- 5%, respectively. These results suggest that PRL is cleaved in a manner that varies with different tissues and physiological states; and thus, forms are produced that might be important mediators of PRL's biological actions on different target tissues. Medium conditioned by preincubation with mammary gland from lactating rats and clarified by 15,000 x g centrifugation had no PRL-cleaving activity at pH 7.4, but gained activity when the pH was lowered, with maximal activity at pH 2.6-3.0. When heated to 85 C for 15 min, such medium had no activity at pH 3.0.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for hepatic involvement in the regulation of amphibian development by prolactin.

Hormonal control of amphibian development involves thyroid hormones (TH), which promote metamorphosis, and prolactin (PRL), which antagonizes the effects of TH and promotes larval growth. Although the liver is not considered to be a regulator of developmental processes such as metamorphosis, it secretes a PRL-synergizing factor (synlactin) in response to PRL. We explored the possibility that the liver may participate in the antimetamorphic actions of PRL in Rana catesbeiana. Bullfrog tadpoles, in which release of endogenous PRL was suppressed by injections of bromocryptine to induce metamorphic changes including tail regression, received hormone-containing implants in various sites. PRL implants in the spleen to deliver hormone directly to the liver via the hepatic portal drainage not only prevented tail regression but actually caused a substantial increase in the height of the tail fin. PRL implanted in other sites or GH implanted in the spleen was much less effective. The liver of animals with intrasplenic PRL implants secreted more synlactin in vitro than that of tadpoles with subcutaneous PRL implants. Young grass frogs were injected with ovine (o) GH or oPRL to determine effects on hepatic synlactin secretion. Although the GH stimulated body growth it did not induce the liver to secrete synlactin. By contrast, PRL treatment did stimulate hepatic secretion of synlactin without stimulating body growth. These results indicate that the liver of pre- and postmetamorphic animals can be stimulated by PRL to secrete synlactin. Furthermore, the antimetamorphic actions of PRL in tadpoles appears to be mediated, at least in part, by an action on the liver. Synlactin may mediate this hepatic effect.