Mapping of syntenic groups U7 and U27 to bovine chromosomes 25 and 12, respectively.
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Animals↗
Biomedical subjects
L Ferretti
Publications and source records attributed to L Ferretti.
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 (7/0.616, 8/0.693, 6/0.641, 5/0.643, 2/0.239, 10/0.844, 6/0.720). The microsatellites were also assigned to synteny groups as follows: IDVGA-2/U17, IDVGA-3/U16, IDVGA-7/U7, IDVGA-8/U29, IDVGA-9/U3, IDVGA-10/U19, IDVGA-11/A (probably U18).
Animals↗
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. Hence, it is concluded that G-banded BTA25 corresponds to R-banded BTA29. The occurrence of other misleading nomenclatures for small bovine chromosomes is discussed.
Animals↗
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. 22.8% had multiple sites for one or more of the rare cutters tested. Sequence analysis for 16 clones confirmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands.
Animals↗
[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.
Aged↗
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. coli of an active preholoenzyme. Furthermore, the reductase precursor was found to undergo a processing in E. coli. The proteolysed form, which retained 13 of the 55 residues of the transit peptide, was active, suggesting that removal of the first 42 residues of the presequence enabled the protein to properly fold and to bind the FAD prosthetic group in the bacterial host, as it was previously shown in the case of the mature form of the spinach reductase.
Amino Acid Sequence↗
Two additional MspI RFLPs revealed by MC.34 (D2S63).
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Chromosome Mapping↗
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.lambda B', starting from 7.5 ng of MC DNA, and describe the isolation of single-copy DNA clones from the library in a two-step procedure. We employed in situ hybridization to unambiguously select the clones specific for the MC, and used three of these clones to demonstrate that it originated from chromosome 9.
Chromosomes, Human, Pair 9↗
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.
Abnormalities, Multiple↗
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. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1.
Animals↗
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. The cloned sequence has been shown to be amplified in cultured rice cells. The analysis of practically intact chromosomal rice DNA molecules by pulsed field gel electrophoresis has now shown that the amplification is associated with the appearance of extrachromosomal molecules. In fact, pE10 hybridizes exclusively with unfractionated DNA from leaf protoplasts, while it recognizes predominantly an extrachromosomal DNA molecule (ECD) of about 45 kb and its multiples in the case of protoplasts from cultured cells. Insensitivity to the action of the exonuclease Bal31 suggests that the molecule is circular. Analysis of restriction endonuclease products with both standard horizontal and pulsed field gel electrophoresis suggest that the extrachromosomal DNA, and its chromosomal counterpart, is composed of tandemly repeated units of about 7 kb. Thus, the smaller extrachromosomal circle should contain 6-7 repeats, while the sequence cloned in pE10 is a subset of this repeat. The extrachromosomal DNA represents about 1% of total rice DNA and its level of amplification is not affected by the different phases of growth in culture.
Cells, Cultured↗
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. Restriction fragment length polymorphisms (RFLPs) specific for the A and B alleles were identified for each of the above enzymes, except for AluI, which has a non-polymorphic site 12bp away from the polymorphic one. We have also found two new polymorphic sites for MboII and TaqI in the non-coding regions. These sites differentiate the A allele into two new variants, named A1 and A2. The RFLP analysis permits the characterization of kappa-casein alleles even in the absence of their expression. This should facilitate selective breeding programmes aimed at increasing the frequency of the kappa-casein B allele whose product improves the cheesemaking properties of milk.
Amino Acid 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%. The YACs show segregative and replicative stability.
Animals↗
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. The new markers were a three-allele BstXI RFLP at D9S5 (PIC = .55) and a six-allele microsatellite, typed by polymerase chain reaction, at D9S15 (PIC = .75). We obtained maximum lod scores of 8.25 between FA and D9S5, 10.55 between FA and D9S15, and 9.52 between D9S5 and D9S15, all at zero recombination. Our results, combined with those reported by other authors, reduce maxlod-1 (maximum lod score minus 1) confidence limits to less than 1.1 cM between FA and D9S5, 1.2 cM between FA and D9S15, and 1.4 cM between D9S5 and D9S15. Linkage disequilibrium with FA was found only for D9S15 when all families were evaluated but was also found for a D9S5/D9S15 haplotype in a subgroup of southern Italian families. We conclude that FA, D9S5, and D9S15 are tightly clustered and that studies of geographically restricted groups may reveal a limited number of mutations responsible for the disease in the Italian population. We present preliminary evidence from pulsed-field gel electrophoresis that D9S5 and D9S15 may be less than 450 kb apart. Linkage disequilibrium between FA and D9S15 suggests that the disease gene may be at an even shorter distance from this marker locus, which therefore represents a very good starting point for cloning attempts.
Alleles↗
Isolation and mapping of a polymorphic DNA sequence (lambda MC.34) on chromosome 2 [D2S63].
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Chromosome Mapping↗
Sequence and functional analysis of a divergent promoter from a cryptic plasmid of Lactobacillus acidophilus 168 S.
We have characterized three of at least five plasmids borne by Lactobacillus acidophilus 168 S. Restriction mapping indicates extensive sequence homology between at least two of them (p1 and p3). We have cloned them in Escherichia coli, and for the smallest (p1) we present the sequence of a region with two divergently arranged promoters which probably share a symmetrical (TTTAAA)-35 box and function efficiently in E. coli cells; an open reading frame contiguous to the promoter, which codes for a 120 amino acid protein of unknown function, and is transcribed in E. coli; and a transcription termination sequence next to this open reading frame. The promoter region contains an AT cluster which is similar to that of the ori2 region of the E. coli F plasmid, and is probably involved in the control of the replication of p1.
Base Sequence↗
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%.
DNA, Bacterial↗
Heterologous expression in Bacillus subtilis. II. In vitro removal of the attenuator sequence of the Escherichia coli his operon allows expression of the cloned hisG gene in B. subtilis.
The promoter-proximal region of the Escherichia coli histidine (his) operon, including the promoter, the attenuator and the hisG gene, as well as the first of the nine structural genes of the his operon, have been cloned in Bacillus subtilis. In this host, the hisG gene could not be expressed because its transcription appeared to be irreversibly terminated at the attenuator (Ferretti et al., 1984). When the attenuator plus various lengths of the two bordering regions were removed, one of the attenuatorless sequences cloned in B. subtilis allowed the progression of transcription and complementation of the corresponding hisA mutation in this Gram-positive host. The deletion removed a 349-bp segment which contained the his attenuator and promoter. In B. subtilis, the productive transcription of the hisG gene started at a site in pAT153 and terminated in pC194. Sequence analysis of the deletion indicates that the E. coli ribosome-binding site of the his operon was used for the translation of the E. coli hisG gene mRNA in B. subtilis cells, which can thus grow in the absence of histidine.
Bacillus subtilis↗