Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)_n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.     

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii

Homoveratric acid (HVA) degradation was observed in cultures of Pleurotus eryngii lacking lignin peroxidase (LiP) activity. Extracellular enzymes seemed responsible for this transformation, and the lack of activity after ultrafiltration of the culture liquid suggests that the presence of some low-molecular-size compounds is required. This hypothesis is supported by rapid HVA transformation after addition of the synthetic laccase substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to the ultrafiltered liquid. HVA transformation by the extracellular enzymes from P. eryngii takes place via C-C breakdown and formation of veratryl alcohol, which is further transformed into veratraldehyde. The same major compounds were found during HVA transformation by LiP from Phanerochaete chrysosporium, but this reaction was not stimulated by ABTS. Although the involvement of other enzymes cannot be ruled out, purified laccase from Pleurotus eryngii caused the same HVA transformation pattern in presence of ABTS. Moreover, veratryl alcohol oxidation by P. eryngii laccase was demonstrated in the presence of ABTS. These results suggest that enzymatic systems lacking LiP could be responsible for natural degradation of lignin.     

A population analysis of Robertsonian and Ag-NOR polymorphisms in brown trout (Salmo trutta)

An analysis of Robertsonian polymorphism and variation in the number of active NORs has been carried out in several populations of brown trout (Salmo trutta) from Northwestern Spain. The karyotype of this species appears to be soundly established, and essentially no variation has been found in chromosome number. Interindividual and interpopulation variation in arm number was detected, with figures ranging between 100 and 102 among individuals, and between 100.10 and 100.80 among populations. This variation in arm number is solely attributable to the polymorphism of the short arm of the main NOR-bearing pair 11, which can appear from acrocentric to metacentric in different individuals. Most populations analyzed showed the standard distribution of active NORs previously observed in this species. The Miño drainage basin, and specially the Chamoso population, showed a multi-chromosomal distribution of active NORs, with several new locations, always telomeric. In most cases no concordance was observed between previously detected rDNA sites in S. trutta and the new Ag-NOR locations. This fact suggests a transposition mechanism rather than an activation of silent rDNA sites to explain this multichromosomal NOR pattern.     

Sucrose transport and hydrolysis inRhizobium tropici

TheRhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN₃) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, thatR. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.     

Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.     

Sperm concentration influences fertilization and male pronuclear formation in vitro in pigs

To study the effect of sperm concentration on the results of pig in vitro fertilization (IVF), 313 oocytes recovered from oviducts of prepubertal gilts after induction of ovulation were used. After capacitation, the number of live spermatozoa in the fertilization dishes was adjusted to 3 x 10(5), 6 x 10(5) and 12 x 10(5) cell/ml. After 4 hours of co-culture in TCM-199, the oocytes were pippeted to remove cumulus cells and the excess spermatozoa around the zona pellucida, and were transferred to fresh TCM-199 for another 12 14 hours . The results showed that 6 x 10(5) spermatozoa/ml is the optimum concentration for this system; the percentage of fertilized ova (71.6%) was not different from the best (76.8%), that was obtained with the highest concentration, and the percentage of monospermy (62.3%) was not different from the best (68.1%), that was obtained with the lowest concentration. The percentage of spermatozoa that reached the pronuclear stage increased while sperm concentration was decreased. The percentage of spermatozoa at the decondensed stage was decreased when the sperm concentration increased.     

Three new HLA-G alleles and their linkage disequilibria with HLA-A

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A T), but conservative, (Thr Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A T) at the third base of codon 107 and (G A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.The nucleotide sequence data reported in this paper have been submitted to the GenBank and EMBL nucleotide sequence databases and have been assigned the following accession numbers: EMBL-X60983 (HLA-G*II), GenBank-M99048 (HLA-G*III), and GenBank-L07784 (HLA-G*IV).The contribution to this paper by P. Morales and A. Corell is equal, and the order of authorship is arbitrary. Correspondence to: A. Arnaiz-Villena.