Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing

The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M_II oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.     

Cell wall alterations in staphylococci growing in situ in experimental osteomyelitis

When cells of both Staphylococcus aureus and Staphylococcus epidermidis are grown in batch culture in nutrient-rich media, their cell walls are regular in thickness, their cell size is within the normal range for each species, and their septation patterns are orderly. When cells of each of these species are examined directly in infected tissue in the rabbit tibia model infection, their cell wall thickness is often much increased and very irregular around the circumference of the cell, their cell size is often increased, and their septation patterns are often severely deranged. All of these alterations in cell wall structure occur in the absence of antibiotics, and we suggest that they may be an expression of phenotypic plasticity in response to altered environmental conditions such as specific nutrient limitations, the presence of antibacterial factors, and growth of the cells on hard surfaces such as rabbit bone or plastic catheters. Some of these specific cell wall alterations are also seen when staphylococcal cells are exposed, in vitro or in vivo, to antibiotics such as clindamycin, but we emphasize that growth in tissue alone is sufficient for their induction.     

Effects of embryo-freezing and thawing techniques on the survivability of Brucella abortus

A suspension of a pathogenic strain (2308) of Brucella abortus was aliquoted, centrifuged, resuspended in 6 treatment media and quantitated. Ten 1-ml samples of each treatment were subjected to a standard embryo-freezing technique. The treatments were selected to examine the effects of 1) freezing and thawing, 2) cryoprotectants and 3) antibiotics on the survivability of Brucella suspended in embryo-support media. Five samples of each treatment were thawed and quantitated after a 2-wk storage period and five samples were thawed and quantitated after a 6-mo storage period. Means and percent reductions were determined for each treatment. There was no statistical difference between means at 2 wk and 6 mo within any treatment. Freezing and thawing caused a 64% reduction in the number of viable Brucella . The addition of antibiotics caused a 99.9% reduction in viability of the organism. Glycerol protected the organism during freezing and thawing in the absence of antibiotics but did not interfere with the high percent reduction seen when antibiotics were present. Dimethylsulfoxide (DMSO), however, not only protected the organism during freezing and thawing but also appeared to negate the effects of the antibiotics.     

Antibiograms of pathogenic bacteria isolated from laboratory rabbits in Ibadan, Nigeria

Antibiotic sensitivity patterns of 21 bacterial isolates from some clinically ill New Zealand rabbits were evaluated against 12 commonly used antibiotics by the Kirby-Bauer disk-diffusion sensitivity testing method. The 21 isolates consisted of six Bordetella bronchiseptica, eight Pasteurella multocida, four Staphylococcus aureus and three Pseudomonas alcaligenes that were associated with snuffles, pneumonia, otitis media, genital infections and conjunctivitis in these groups of caged rabbits. The four bacteria species were all sensitive to kanamycin, gentamycin and enrofloxacin, with variable sensitivity to the other antibiotics tested. The results of this antibiogram could serve as a field guide in the treatment of very acute bacterial diseases of rabbit prior to the availability of the results of local sensitivity tests. Such sensitivity tests should be reviewed yearly to update this antibiogram.     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.     

Campylobacter and fluoroquinolones: a bias data set?

There is no universally accepted standard method for the isolation of Campylobacter spp. and it is considered that currently available isolation media are not yet optimal for the recovery of Campylobacter spp. from a range of sample types. Almost all methods incorporate antibiotics into the isolation media to inhibit growth of other bacteria within the sample. It is established that the incorporation of such antibiotics into isolation media will inhibit the growth of some Campylobacter spp. as well as other bacteria. The results of the use of such suboptimal isolation methods are that the isolates which 'survive' the isolation procedure will be those which: (i) are able to 'out compete' the rest of the bacteria in the sample, i.e. they are able to grow faster; (ii) are resistant to the antibiotics used in the isolation media; and (iii) are randomly selected by the laboratory technician as being a 'typical'Campylobacter spp. It is clear that such a procedure is intrinsically biased and will mean that species resistant to the antibiotics used in the media will be isolated. This introduces real doubt that the bacteria isolated are truly representative of those initially found on the sample. It is also becoming clear that Campylobacter spp. are rather difficult to isolate as pure cultures and many are in fact mixtures of more than one strain. Again this introduces great uncertainty as to the prevalence and distribution of respective species from the different sample types. This is especially true when considering isolation of Campylobacter spp. causing disease in man as there is no certainty that the selected isolate is that which was responsible for disease. The incorporation of antibiotics into the isolation media not only introduces the issue of species bias but perhaps more importantly exposes the Campylobacter spp. to a cocktail of antibiotics thereby providing the potential for them to 'switch on' antibiotic resistance mechanisms. It might be argued that this has always been the case for isolation of Campylobacter spp., however, we know that the antibiotic cocktails used in media over the last 10 years have changed and indeed there was a time when the filtration protocol which didn't use antibiotics was more widely used. As most reports in the literature do not state what methods were used to isolate Campylobacter spp. it is not possible to quantify any relationship between antibiotics used in the isolation media and susceptibility data. An approved method for Campylobacter susceptibility testing was not available until May 2002, all data generated prior to this date will have been generated using non-standard methods. As tremendous variability in the reproducibility data for Campylobacter spp. was observed during the development of the standard agar dilution susceptibility method, data generated with disk diffusion and broth microdilution methods must be considered with caution. It has been shown that, compared with the conventional agar dilution method, the E-test tends to give rise to lower minimal inhibitory concentrations (MICs) for sensitive strains and higher MICs for resistant strains. There are no recommended antibiotic breakpoint concentrations for Campylobacter spp. A breakpoint is used to separate sensitive from resistant strains of bacteria and is thus crucial to any discussion of antibiotic resistance. This discussion is further complicated by introduction of the terms microbiological and clinical breakpoints. While a microbiological breakpoint can be a useful parameter with regard to identifying resistance factors it cannot on its own be used to predict whether that bacteria will respond to treatment from an appropriate antibiotic. Predicting clinical response is a function of the clinical breakpoint which considers the pharmacokinetic profile of the antimicrobial compound, i.e. the concentration of the antimicrobial compound in the body and the MIC. The National Committee for Clinical Laboratory Standards (NCCLS) uses microbiological, pharmacokinetic and clinical data to establish breakpoints, without c and clinical data to establish breakpoints, without such considerations it is not possible to consider what is truly clinically sensitive and resistant. There are no reported studies that have systematically determined appropriate breakpoints for Campylobacter, there are data however, which relate MICs to clinical outcome. It is without dispute that microbiological resistance in Campylobacter spp. occurs as a result of mutation in the gyrA gene with single point mutations most frequently causing a four- to eightfold shift in the MIC. What is also clear is that if a high enough concentration of antimicrobial relative to MIC of the infecting organism can be achieved not only will the parent organism be killed but also the 'resistant' mutant. Considering the above and the concentrations of ciprofloxacin achieved in the gastro-intestinal tract it is not surprising that clinical cure can be demonstrated for organisms with an MIC of 32 microg ml(-1).     

Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Genetic Control of Mitochondria in Paramecium

Mitochondrial mutations for resistance to various antibiotics (erythromycin, chloramphenicol, spiramycin, mikamycin) have been obtained in Paramecium aurelia and their properties are reviewed. Using these mitochondrial markers, the interactions between nucleus and mitochondria have been studied in two ways: by microinjection of mitochondria from one stock or species into other stocks and species of P. aurelia and by a genetic study of a nuclear mutation affecting mitochondrial multiplication. Both types of experiments show: (1) that there may exist incompatibility between a given type of mitochondria and the cell into which they are introduced and (2) that through multiplication in the host cell, mitochondrial properties can be modified. The possible basis for incompatibility and host-induced modifications is discussed.     

6-Phosphofructo-1-kinase isoenzymes of the jejunal mucosa of rabbit, rat and mouse

1. 6-Phosphofructo-1-kinase (PFK) isoenzymes were studied in the jejunal mucosa of rabbit, rat and mouse. 2. The rat mucosal enzyme was found to be very similar to, although not identical with, the mouse mucosal enzyme, as the physical and regulatory properties of these two enzymes were nearly similar except that the immunological studies were dissimilar. 3. PFK prepared from rabbit mucosa showed different and distinct properties from the rat and mouse mucosal PFK when studied by (NH4)2SO4-precipitation, polyacrylamide gel electrophoresis, immunological cross-reactivity and regulatory properties. 4. The difference between the rabbit enzyme and the rat or mouse enzymes is suggested to be due to the lower rate of glycolysis observed in the rabbit jejunal mucosa as the total enzyme activities of the rabbit were found to be less than half of those activities of the rat and mouse mucosa. 5. The dissimilarities among the species in mucosal isoenzymes obtained in the present study are rather expected since the term isoenzyme is now properly reserved for forms that have been shown to be genetically distinct as shown for different tissues in the same species. Such multigenic control does not appear to have been established for the same tissue in different species.     

Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis.     

Plasmid transfer and susceptibility to antibiotics in the halophilic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens

The present study defines a series of genetic procedures to be used for molecular studies in photosynthetic halophilic species such as Rhodovibrio salinarum and Rhodothalassium salexigens. In both species, the minimal inhibitory concentrations for the antibiotics tetracycline, rifampicin, chloramphenicol, spectinomycin, streptomycin, and kanamycin were determined. In addition, conjugal transfer of IncP and IncQ plasmids from Escherichia coli was demonstrated and the resistance markers expressed in these halophiles were determined. Finally, Rth. salexigens growth dependence on variable salt concentrations was measured: maximal growth rates were seen at 6% and 4% NaCl under phototrophic and chemotrophic conditions, respectively. To the best of our knowledge, this is the first report analyzing the genetic properties of two representative species of halophilic purple non-sulfur phototrophs.     

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.     

Rabbits – their domestication and molecular genetics of hair coat development and quality

The European rabbit (Oryctolagus cuniculus) is the only representative of its genus living in present‐day Europe and North Africa, and all domestic rabbits are descendants of this one species, which is native to the Iberian Peninsula. There are over 300 breeds of rabbits that differ in size, coat color, length of ears and type of fur. Rabbits are bred for various reasons, such as for laboratory animals and a source of meat, wool and fur, as well as for pets and exhibition animals. The hair coat is a important economic trait of rabbits. Its development and quality are influenced by various factors, both environmental and genetic. The genetic mechanisms underlying its development have not been thoroughly researched. The aim of this review is to discuss the domestication of rabbits and the different aspects of rabbit genetics. A brief review of the properties of rabbit hair coat, hair coat development and hair cycle will be provided, followed by discussion of the factors regulating hair coat development, molecular control of hair coat development and the role of non‐coding RNAs in the regulation of gene expression in the hair follicles of rabbits. Information about genetic regulation of pathways could provide useful tools for improving hair coat quality and be of practical use in rabbit breeding.     

Genetic influences on behavioral inhibition and anxiety in juvenile rhesus macaques

In humans and other animals, behavioral responses to threatening stimuli are an important component of temperament. Among children, extreme behavioral inhibition elicited by novel situations or strangers predicts the subsequent development of anxiety disorders and depression. Genetic differences among children are known to affect risk of developing behavioral inhibition and anxiety, but a more detailed understanding of genetic influences on susceptibility is needed. Nonhuman primates provide valuable models for studying the mechanisms underlying human behavior. Individual differences in threat-induced behavioral inhibition (freezing behavior) in young rhesus monkeys are stable over time and reflect individual levels of anxiety. This study used the well-established human intruder paradigm to elicit threat-induced freezing behavior and other behavioral responses in 285 young pedigreed rhesus monkeys. We examined the overall influence of quantitative genetic variation and tested the specific effect of the serotonin transporter promoter repeat polymorphism. Quantitative genetic analyses indicated that the residual heritability of freezing duration (behavioral inhibition) is h ² = 0.384 ( P  = 0.012) and of 'orienting to the intruder' (vigilance) is h ² = 0.908 ( P  = 0.00001). Duration of locomotion and hostility and frequency of cooing were not significantly heritable. The serotonin transporter polymorphism showed no significant effect on either freezing or orienting to the intruder. Our results suggest that this species could be used for detailed studies of genetic mechanisms influencing extreme behavioral inhibition, including the identification of specific genes that are involved in predisposing individuals to such behavior.     

Phytoremediation potential of Myriophyllum aquaticum and Pistia stratiotes to modify antibiotic growth promoters, tetracycline, and oxytetracycline, in aqueous wastewater systems

Antibiotics are frequently used in the United States as feed efficiency promoters and medicines for livestock that is destined for human consumption. These antibiotics are released into the environment through the runoff and wastewater streams from animal feedlots and land applications of manure. The exposure of microorganisms to these antibiotics has reportedly resulted in the development of resistant species of microorganisms, which in turn can lead to human health hazards. Phytoremediation of these antibiotics can be a useful tool for countering this problem. Aquatic plants, Myriophyllum aquaticum (parrot feather) and Pistia stratiotes (water lettuce), were used for studying phytoremediation of tetracycline (TC) and oxytetracycline (OTC) from aqueous media. TC and OTC are two of the most commonly used tetracyclines in veterinary medicine. M. aquaticum and P. stratiotes gave high antibiotic modification rates of both antibiotics. Kinetic analyses dismiss direct enzyme catalysis; the modification rates decreased with increasing OTC concentrations. Sterile, cell-free root exudates (filtered through 0.2 microm membranes) from both species also exhibited comparable antibiotic modification rates. The involvement of root-secreted metabolites in antibiotic modification is suggested. The changes in the UV absorbance spectra of OTC during treatment with the root exudates confirmed the modification.     

Media alternatives for the collection, culture and freezing of mouse and cattle embryos

The replacement of biological products in media for the collection, culture and freezing of mammalian embryos was studied. To test the hypothesis that chemically defined surfactants can replace bovine serum albumin (BSA) or serum in embryo media, morula-stage mouse and cattle embryos were collected, cultured, and/or frozen in the surfactant compound, VF5. Collection efficiency of mouse and cattle embryos did not differ whether the medium contained serum or surfactant. In addition, morula-stage mouse and cattle embryos developed and hatched at similar rates in culture media containing either BSA or surfactant. Although the freeze/thaw survival and development in culture of bovine embryos was not significantly different in any of the media, there was a significantly lower hatching rate of mouse embryos frozen with serum or surfactant than with cryoprotectant alone or with cryoprotectant plus albumin-free serum. However, the absence of serum or surfactant in embryo freezing media resulted in embryo loss, presumably due to stickiness. The data suggest that serum can be replaced by a chemically defined surfactant in mouse and cattle embryo transfer systems for the collection, culturing and freezing of embryos. It is likely that the beneficial effects of serum are due to its surfactant properties.     

Antibiotic resistance in bacterial isolates from laboratory animal colonies naive to antibiotic treatment

Antiobiogrammes were made of a number of isolates of Staphylococcus aureus, Escherichia coli and Pasteurella pneumotropica derived from rodent, rabbit or minipig colonies never treated with antibiotics. For S. aureus no differences between rats and mice were found in the percentage of resistant isolates. Gentamicin and erythromycin were found to be the most efficient, while the highest percentages of resistance were found to be against penicillins and sulphonamides. In general, the results from antibiogrammes on E. coli were rather uniform, with only slight differences between isolates from different species, except that more vancomycin and tetracycline-resistant minipig isolates were found. In almost all isolates of E. coli, resistance was shown against penicillin, fucidin, macrolides, lincosamides and tiamulin. For a number of antibiotics, mouse isolates of P. pneumotropica were more frequently found to be sensitive than rat isolates. The resistance patterns of E. coli from the minipigs were quite similar to resistance patterns found in farm pigs, but apart from this, the resistance patterns of the bacterial species tested did not resemble human or farm animal patterns in any of the animal species, and, therefore, these studies do not support the theory that S. aureus and E. coli in laboratory animal colonies derive from the normal flora of the human caretakers. The fact that rodent species of E. coli, in contrast to human and farm animal species, are sensitive to ampicillin, tetracyclines, and the combination of sulphonamides and trimethoprim, might be due to the fact that these antibiotics are not used in rodent populations.     

The development of gene expression systems for filamentous fungi

Filamentous fungi have been used for decades in the commercial production of enzymes, antibiotics, and specialty chemicals. Traditionally, improving the yields of these products has involved either mutagenesis and screening or modification of fermentation conditions. Generally, selective breeding of strains has not been successful, because most of the commercially important fungal species lack a sexual cycle. For a few species, strain improvements have been made possible by employing the parasexual cycle for genetic crosses (30). The recent development of DNA-mediated transformation systems for several industrially important fungal species has spawned a flurry of research activity directed toward the development of gene expression systems for these microorganisms. This technology is now a viable means for novel and more directed approaches to improving existing fungal strains which produce enzymes or antibiotics. In addition, fungal expression systems are now being tested for the production of heterologous gene products such as mammalian pharmaceutical proteins. The goal of this review is to present a summary of the gene expression systems which have recently been developed for some filamentous fungi of commercial importance. To insure that the most recent developments are presented we have included data from not only scientific papers, but also from personal communications, abstracts, symposia, and our own laboratory.