Six antimicrobial peptide genes of the cathelicidin family map to bovine chromosome 22q24 by fluorescence in situ hybridization.

Six phage clones containing gene members of the family of antimicrobial peptides named cathelicidins, were mapped to bovine chromosome 22q24, by means of fluorescence in situ hybridization. The mapping data suggest the clustering of cathelicidins into a CATHL@ locus, in a similar manner as for beta-defensins, another family of antimicrobial peptides, defining the locus DEFB@ mapped to 27q13-->q14.

T-banding pattern of bovine chromosomes and karyotype reconstitution with physically mapped cosmids.

Bovine T-banded chromosomes were identified by fluorescence in situ hybridization (FISH) using 27 cosmid probes as chromosome landmarks. Pairwise combinations of probes from chromosomes of markedly different size were cohybridized to metaphase spreads of T-banded chromosomes. This confirmed the association of banding patterns to individual chromosomes.

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites.

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap.

Presence of a chloroplast DNA sequence in an autonomous circular DNA molecule in cultured rice cells (Oryza sativa L).

Sequence analysis of twelve DNA fragments, which had previously been found to be extensively amplified in suspension-cultured rice cells, revealed that two of them, isolated on plasmids designated pE10 and pE11, have sequences identical to distinct regions of chloroplast DNA (ct-DNA). Both sequences are part of an extrachromosomal circular DNA molecule (ECD). The molecular structure of the ECD was investigated by a combination of restriction analysis, standard and pulsed-field gel electrophoresis, hybridization with ct-DNA probes and amplification by the polymerase chain reaction in the presence of oligonucleotide primers homologous to selected regions of rice ct-DNA.

Combined Q-banding and fluorescence in situ hybridization for the identification of bovine chromosomes 1 to 7.

Eleven probes were assigned to bovine chromosomes 1 to 7 by fluorescence in situ hybridization (FISH). The identification of chromosomes was based on QFQ-banding prior to in situ hybridization and comparison with the Reading Conference (1976) and ISCNDA (1989) standards. The probes used for FISH can now be utilized as identification and discrimination features for bovine chromosomes 1 to 7 and particularly for chromosomes 4 and 6, which are difficult to distinguish.

Direct characterization of bovine microsatellites from cosmids: polymorphism and synteny mapping.

A (TG)8 oligonucleotide probe was used to screen 186 cosmids from a commercial bovine cosmid library. Of the 56 positive discovered, 7 were sequenced in the region of the microsatellite and analysed for polymorphism. These microsatellites, IDVGA-2, -3, -7, -8, -9, -10, -11 showed the following number of alleles and polymorphism information content (PIC) values (7/0.

Bovine synteny group U7, previously assigned to G-banded chromosome 25 in the ISCNDA nomenclature, assigns to R-banded chromosome 29.

Four microsatellite-containing bovine cosmids have been regionally localized by fluorescence in situ hybridization to bovine R-banded chromosome 29 (BTA29) and to the 1;29 translocated chromosomes. PCR-analyses of a somatic hybrid cell panel assigned the four microsatellites to synteny group U7. One of the cosmids (IDVGA7) has been previously mapped to G-banded BTA25 allowing to assign U7 to this chromosome.

Construction of a library of bovine genomic fragments enriched in CpG islands.

A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endonucleases. A collection of SacII (CvCGCGG)-Hin-dIII fragments from bovine sperm was established in the plasmid Bluescript. 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters: EagI, BsshII, NarI, MluI, NruI, NaeI: 70% of the clones contained at least 1 site and 5% contained four different such sites.

[The changes in the sexual behavior of patients undergoing aorto-iliac revascularization].

A correct approach to sexual disorders in vascular patients presupposes an accurate investigation before and after operation. The authors evaluate the reliability of the diagnostic methods used, pre- and postoperatively, to ascertain sexual disorders, and analyse the influence of surgery on sexual function.

Studies on the holoenzyme biogenesis of the spinach ferredoxin-NADP+ reductase.

An expression plasmid, pPreFNR, in which the DNA sequence coding for the spinach ferredoxin-NADP+ reductase precursor was under the control of prokaryotic transcription and translation initiation signals has been constructed. The plasmid directed the synthesis in Escherichia coli of a 43-kDa immunoreactive polypeptide which could be identified with the reductase preprotein. Analyses of bacterial extracts showed that the precursor was unstable and devoid of catalytic activities, suggesting that the presence of the transit peptide would not allow the assembly in E.

Preparation of high molecular weight plant DNA and its use for artificial chromosome construction.

The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension.

Molecular cloning of DNA from a sorted human minichromosome.

A human supernumerary minichromosome (MC), found in a newborn baby and sorted on a fluorescence-activated cell sorter (FACS-440) has been previously described [Ferretti et al., Cytotechnology 1 (1987) 7-12]. We report here on the construction of a library of EcoRI fragments in the phage lambda gtWES.

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

A supernumerary minichromosome has been detected in a severely malformed patient. Attempts at identifying the marker by conventional approaches were unsuccessful. The physical isolation of the minichromosome by fluorescence activated sorting, molecular cloning of its DNA, and in situ hybridisation experiments performed with single copy DNA probes allowed us to show that it was derived from a rearrangement involving the centromere and the proximal region of the short arm of chromosome 9.

Long range restriction analysis of the bovine casein genes.

Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes.

Friedreich ataxia in Italian families: genetic homogeneity and linkage disequilibrium with the marker loci D9S5 and D9S15.

Friedreich ataxia (FA) is an autosomal recessive degenerative disease of the nervous system of unknown biochemical cause. The FA gene has been shown to be in close linkage with the two chromosome 9 markers D9S5 and D9S15, and linkage disequilibrium between FA and D9S15 has been detected in French families by Hanauer et al. We used new highly informative markers at the above loci to analyze Italian FA families for linkage and linkage disequilibrium.

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.).

The plasmid pE10 is a pBR322-derived plasmid carrying a 4.5 kb rice (Oryza sativa L.) repeated DNA sequence.

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%.

Restriction fragment length polymorphism analysis of the kappa-casein locus in cattle.

The two common genetic variants (A and B) of bovine kappa-casein originate from two point mutations in the codons for the aminoacids in position 136 and 148. These mutations give rise to polymorphic sites for the restriction endonucleases Hin dIII, AluI, HinfI, Mbo II and TaqI. We have examined DNAs of several Italian Friesian cows and bulls of known and unknown genotype by Southern analyses using kappa-casein cDNA probes.

Cytological analysis and sorting of a human supernumerary minichromosome.

In a newborn female, an abnormal karyotype, 46,XX/47,XX,+mar/47,XX,+9, was found associated with several malformations. The marker chromosome was present in 70% of peripheral blood lymphocytes, and its size appeared to be less than half of the smallest chromosomes. Several differential staining methods provided no indication as to its origin.

Lactobacillus protoplast transformation.

A method for the transformation of Lactobacillus protoplasts by plasmid DNA is reported. The procedure involves polyethylene glycol treatment of protoplasts to induce DNA uptake. A transformation efficiency ranging from 5 to 1000 transformants per microgram of DNA is achieved; the efficiency of protoplast regeneration ranged from 10 to 20%.