In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterne. The cytodifferentiation of the oviduct cells was induced by 17β-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.     

Avidin induction by tissue injury and inflammation in male and female chickens.

1. The occurrence and inducibility of the biotin-binding egg white protein (avidin) in the chicken is not restricted to the oviduct. 2. Inflammatory treatments (intestinal injury, actinomycin D) induced avidin in a number of tissues of young and adult hens and roosters, but not of female rats and mice. Highest avidin concentrations were found in the organs containing epithelial cells and serous membrane. 3. The expression of the avidin gene by tissue injury and inflammation suggests that avidin has a significant function in the injured and inflamed chicken tissues.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Variants of ST8SIA1 are associated with risk of developing multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios (affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.     

Carbonic anhydrase inhibitors: the very weak inhibitors dithiothreitol, beta-mercaptoethanol, tris(carboxyethyl)phosphine and threitol interfere with the binding of sulfonamides to isozymes II and IX

The inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with dithiothreitol, 2-mercaptoethanol, tris(carboxyethyl)phosphine (reducing agent frequently added to enzyme assay buffers) and threitol has been investigated. The agents were very weak inhibitors of isozymes CA II and CA IX, but unexpectedly, strongly influenced the binding of the low nanomolar sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide). Acetazolamide affinity for all investigated CAs diminished orders of magnitude with increasing concentrations of these agents in the assay system. DTT and similar derivatives should not be added to the assay buffers used in monitoring CA activity/inhibition, as they lead to under-estimation of the binding constants, by a mechanism probably involving the formation of ternary complexes.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Ratio-dependent parasitism with Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) on egg rafts of Nezara viridula (Linnaeus) (Hemiptera: Pentatomidae): effect of experimental variables and compatibility of 'ratio' and 'Holling' models

Abstract Egg rafts of Nezara viridula were exposed to the parasitoid wasp Trissolcus basalis in experimental arenas to establish the relationship of the rates of attack and parasitism to various combinations of arena size, parasitoid density, host density and parasitoid-to-host ratio. Arena sizes were varied in the ratio 1:9:63, with the largest having a search area of 1.44 m². Parasitoid and host densities were varied over a 27-fold range. The parasitoid-to-host ratios used were 1:1, 3:1 and 6:1. Finding time was related to a constant factor (flight propensity), rather than to the difficulty of finding (density of hosts). Initial attack rates were therefore related only to parasitoid numbers (or density), even at the lower densities and ratios. Parasitism rates (a function of attack rate per host) were thus also strongly related to parasitoid to host ratio, regardless of densities used and arena sizes. Even reducing host density, while keeping time and parasitoid density constant, increased the parasitism rate. A ratio model for parasitism rate was therefore compatible with the data but the more explicit Holling 'disc' equation was also compatible because handling time was sufficiently large to make it sensitive to the ratio of parasitoids to hosts for the densities used. We conclude that the two models would predict different results if the density of host egg rafts was in a range below one per square metre.