Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then metabolized to 2,5-dichlorohydroquinone. Here, we isolated from the genomic library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when they were transformed into P. putida and Escherichia coli. Both gene products had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first gene, named linC, located separately from the two genes (linA and linB) which we had already cloned as genes involved in the gamma-HCH degradation. The other, named linX, located about 1 kb upstream of the linA gene encoding gamma-HCH dehydrochlorinase. A gamma-HCH degradation-negative mutant, named UT72, which lacked the whole linC gene but had the intact linX gene was isolated. The linC gene given in a plasmid could complement UT72. These results strongly suggest that the linC gene but not the linX gene is essential for the assimilation of gamma-HCH in UT26. Deduced amino acid sequences of LinC and LinX show homology to those of members of the short-chain alcohol dehydrogenase family.     

Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants

Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol (DEG) or 1.3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline. (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0.25-ml plastic straws and were placed directly into a 0 degrees C alcohol bath chamber and held for 2 min. They were cooled from 0 degrees C to -5.5 degrees C at 1 degrees C/min and then seeded, followed by a 10-min holding period at -5.5 degrees C. The straws were then cooled to -30 degrees C at 0.3 degrees C/min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05), between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.     

Axillary bud flowering after apical decapitation in Pharbitis in relation to photoinduction

The flowering response of axillary buds of seedlings of Pharbitis nil Choisy, cv. Violet, was examined in relation to the timing of apical bud removal (plumule including the first leaf or second leaf) before or after a flower-inductive 16-h dark period. When the apical bud was removed well before the dark period, flower buds formed on the axillary shoots that subsequently developed, but when removed just before, or after, the dark period, different results were observed depending on the timing of the apical bud removal and plant age. In the case of 8-day-old seedlings, fewer flower buds formed on the axillary shoots developing from the cotyledonary node when plumules were removed 20 to 0 h before the dark period. When the apical bud was removed after the dark period, no flower buds formed. Using 14-day-old seedlings a similar reduction of flowering response was observed on the axillary shoots developing from the first leaf node when the apical bud was removed just after the dark period. To further elucidate the relationship between apical dominance and flowering, kinetin or IAA was applied to axillary buds or the cut site where the apical bud was located. Both chemicals influenced flowering, probably by modulating apical dominance which normally forces axillary buds to be dormant.     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.     

Variable X chromosome inactivation patterns in near-tetraploid murine EC × somatic cell hybrid cells differentiatedin vitro

For the cytogenetic study of X chromosome inactivation as an X chromosome dosage compensation mechanism, we isolated a number of XXXX, XXX, and XXY near-tetraploid mouse hybrid cell clones by fusing XX or XO embryonal carcinoma cells with lymphocytes carrying a structurally altered X chromosome(s). The inactive X chromosome from the female lymphocyte was reactivated in these hybrid clones which retained embryonal carcinoma morphology so far as they were cultured on the collagen-coated plastic surface in the medium supplemented with leukemia inhibitory factor (LIF) and betamercaptoethanol (BME). Some of these clones developed balloon-like cystic embryoid bodies when they were allowed to form cell aggregates in medium without LIF and BME in bacteriological petri dishes to which they do not adhere. X chromosome inactivation occurring during this process detected by the incorporation of 5-bromodeoxyuridine did not conform to the expected pattern leaving two X chromosomes active in every tetraploid cells. This may suggest either that the X-inactivation mechanism evolved primarily, for the diploid cell is unable to deal with tetraploid conditions efficiently, or that the present system ofin vitro differentiation represents an anomalous situation never encounteredin vivo.     

Development of the Overlapping Oligonucleotide Database and its application to signal sequence search of the human genome

We have developed ODS (Overlapping Oligonucleotide Databasefor Signal Sequence Search)—the first relational databasethat integrates information on biological features into thesearch for signal sequences. In existing biological sequencedatabases, even relational ones, retrieving nucleotide sequencesbased on their biological features involves much labour andtime or even the development of a new program. GenBank sequencedata, including FEATURES records, are organized into three relationaltables in ODS. Nucleotide sequences are transformed into overlappingoligonucleotides in order to facilitate the signal sequencesearch rapidly without the need for specific alignment programs.This transformation leads to a one-to-one correspondence betweenthe nucleotide sequence and its biological feature. The signalsequence search by ODS is done in SQL queries and ODS obviatesthe need for molecular biologists to write computer programs.The application of ODS to searches of promoter regions revealedputative cis-acting elements and basic statistical analysesof occurrences of oligonucleotides showed interesting findingsconcerning the ‘cg’ dinucleotide.     

A deductive database system PACADE for analyzing 3-D and secondary structures of protein

We have developed a deductive database system PACADE for analyzing3-D and secondary structures of protein. The PA CADE systemconsists of a relational database created from Protein DataBank and a deductive engine DEE based on logic programming.It has the following features: (1) The system has an inferencemechanism. This means by which users can easily write and checkbiological hypotheses using logical and declarative rules insteadof procedural programs. (2) The relational database of the PACADE system stores data on bath 3-D and secondwy structuresof protein. The integration of this two level structure makesfeasible an abstract representation of the protein structure.We describe herein the design, functions, and implementationof this PACADE system.