Characterization of the apical membrane antigen-1 in Italian strains of Babesia bigemina.

Babesia bigemina is a parasite endemic in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a trans-membrane antigen recently identified. In this research, we characterized the ama-1 gene from three Italian B. bigemina strains, two B. bigemina strains obtained from Ragusa, Sicily (ITA1 and ITA3) and a third one obtained from Benevento, Campania (ITA2). Italian sequences were compared with those of the Australian strain obtained from the Sanger Institute web site and to strains from different parts of the world. The results obtained confirmed that this newly described ama-1 gene is highly conserved among Italian and foreign strains which has implications for vaccine development.

Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous histotype.

BACKGROUND: K-ras mutations, one of the earliest events observed in colorectal carcinogenesis, are mostly found in codons 12 and 13, and less frequently in codon 61, all three of which are estimated to be critical for the biological activity of the protein. Nevertheless the prognostic significance of such mutations remains controversial. Our purpose was to assess whether any or specific K-ras mutations in primary colorectal cancer had prognostic significance and were linked to clinico-pathological parameters. PATIENTS AND METHODS: Paired tumor and normal tissue samples from a consecutive series of 160 untreated patients (median of follow up 71 months), undergoing resective surgery for primary colorectal carcinoma, were prospectively studied for K-ras mutations by PCR/single strand conformation polymorphism sequencing. RESULTS: Seventy-four of the 160 (46%) primary colorectal carcinomas presented mutations in K-ras: 54% in codon 12, 42% in codon 13 (particularly G-->A transition) and 4% in both. Codon 12 K-ras mutations were associated with mucinous histotype (P <0.01), while codon 13 K-ras mutations were associated with advanced Dukes' stage (P <0.05), lymph-node metastasis (P <0.05) and high S-phase fraction (P <0.05). Multivariate analysis showed that codon 13 K-ras mutations, but not any mutation, were independently related to risk of relapse or death. CONCLUSIONS: Our results suggest that codon 12 K-ras mutations may have a role in the mucinous differentiation pathway, while codon 13 mutations have biological relevance in terms of colorectal cancer clinical outcome.

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases.

Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c-Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0.81; P<0.01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations (P=0.05 and P=0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.

Direct identification of each specific mutation in codon 12 and 13 of ci-ki-ras2 by SSCP analysis.

We compared the SSCP behaviour of the DNA fragments containing c-ki-ras 2 wild type 12 and 13 codons or each of the 12 possible point mutated sequences in these two codons. We found that a single electrophoresis condition was sufficient to distinguish each specific mutation from the other 11 and from the wild type sequence. This observation makes it possible to identify each specific mutation directly by SSCP without any need for reamplification and sequencing.

Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA.

Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions.

Structural analysis of c-myc in human sporadic colorectal carcinomas.

Analysis of the DNA extracted from the colonic mucosa of 13 patients operated for colorectal carcinoma revealed c-myc rearrangements in three of them. In all cases, the alterations were found both in the histologically normal and in the tumoral mucosa. In one case, a different pattern of rearrangements characterized the two tissues. In all three individuals, the c-myc sequences appeared to be amplified. We suggest that c-myc rearrangements in the histologically normal colonic mucosa of colorectal carcinoma patients could represent a predisposition factor for the development of additional neoplasms.

Optimization of RNA and DNA purification procedures from human colorectal carcinomas.

We optimized procedures to extract RNA and DNA separately from colorectal carcinomas pulverized by Microdismembrator II. Our procedures make it possible to achieve good yields and high degree of purification of both types of macromolecules without affecting their structural integrity.

Readthrough transcription occurs at the rho dependent signal F1 TIV in suppressor cells.

Suppressor cells infected with bacteriophage f1 yield phage encoded gene IV transcripts longer than those present in the supo host and identical to those found in a rho- host. However, such longer transcripts do not appear in the suppressor-infected cell when, by changing the translation frame of gene IV, the ribosome is not allowed to proceed to the end of the gene IV message and thus to reach the rho dependent transcription terminator f1 TIV. This suggests that ribosome movement beyond the natural gene IV stop codon disturbs the activity of that termination signal. In contrast to the rho- behaviour, the suppressor does not accumulate high levels of gene IV messages indicating that the accumulation occurring in the rho- mutant may not be a primary effect of the readthrough per se.

Isolation and characterization of a sea urchin hsp 70 gene segment.

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.

Rho-dependence of the terminator active at the end of the I region of transcription of bacteriophage f1.

Infection of rho- Escherichia coli cells with deletion mutant PII of bacteriophage f1 results in a miniphage RNA population composed of transcripts longer than those synthesized in the infection of rho+ cells. This indicates a Rho dependence of the terminator active at the end of the I region of transcription of bacteriophage f1. An estimate of the length of a transcript, which represents a good fraction of the RNA that passes beyond the terminator, indicates that the hairpin structure where synthesis of complementary strand DNA initiates also acts as a fairly efficient Rho-independent terminator.

Transcription in bacteriophage f1-infected Escherichia coli: RNA synthesized on DNA of deletion mutant PII shows the existence of a two-site terminator.

Two different transcripts are synthesized on the DNA of deletion mutant PII of bacteriophage f1 in E. coli cells infected with this miniphage. Both RNA species appear to be primary transcripts and differ by about 100 nucleotides at their 3'OH end. Mapping of these molecules on the miniphage genome suggests that a two-site terminator is active at the end of the I region of transcription of bacteriophage f1.

Transcription in bacteriophage f1-infected Escherichia coli. Messenger populations in the infected cell.

Transcription of bacteriophage f1 DNA in vivo occurs in two independent regions. They are separated from one another by a strong terminator just downstream from gene VIII on one side, and by the filamentous phage intergenic space on the other. One of these regions contains genes II, V, VII, IX and VIII, and is actively transcribed. In this region there are a number of promoters but only one effective terminator. Thus, most of the RNAs that come from this region overlap and share sequences close to the termination site. The other region, which contains genes III, VI, I and IV, is transcribed much less actively. This region gives rise to a long (approximately 4 X 10(3) bases) RNA that covers the entire region, and several RNAs that overlap in the region closest to their 5' termini. Several other RNAs appear to overlap only with the 4 X 10(3) base transcript. Thus, not only the frequency but the organization of transcription differs in the two portions of the genome.

Transcription in bacteriophage f1-infected Escherichia coli: very large RNA species are synthesized on the phage DNA.

Fractionation of pulse-labeled RNA extracted from E. coli cells infected with phage f1 and hybridization of this RNA to f1 DNA reveals that very large species are synthesized on the phage genome. Hybridization of the RNA to specific fragments of f1 DNA shows that, in the infected cell, at least one mRNA is present into which the sequences of genes III, VI, and I are all transcribed together. This result fully explains the polar effect shown by gene III mutants on the expression of genes VI and I (Pratt et al. 1966).