Novel avian leukosis virus-related endogenous proviruses from layer chickens: characterization and development of locus-specific assays.

During the course of evolution, vertebrate genomes have been invaded and colonized by retroviruses. In humans, for example, endogenous retroviruses (long terminal repeat elements) occupy roughly twice as much sequence space as essential genes. There are numerous reports in the literature implicating endogenous proviruses in the modulation of host physiology. The fact that many of these host-virus interactions take place in a proviral locus-specific manner speaks to the need for rapid assays for element profiling. This report deals with the identification of novel elements belonging to a family of endogenous retroviruses, designated ALVE, that reside in the genome of the chicken and that are closely related to exogenous avian leukosis viruses. The study of ALVE elements in the chicken genome serves as a model system for understanding the interplay between endogenous viruses and their vertebrate hosts in general, including humans. In this report, we present locus-specific, diagnostic PCR-based assays for 2 novel ALVE elements. In addition, we characterize the proviral structures and examine the genomic environments of both novel elements along with a previously described element known as ALVE-NSAC-3.

Glucose Repression of Amylase Gene Expression in DROSOPHILA MELANOGASTER.

We have previously shown that dietary glucose can reduce amylase activity in both adults and larvae of Drosophila; this reduction in enzyme activity reflects a reduction in the quantity of amylase protein, rather than an inhibition of enzyme activity. Here, we report that we have now defined conditions in which the repressive effect of glucose can be greater than 100-fold. Moreover, this repression is partially counteracted by the addition of exogenous cyclic AMP. We also show that there is a direct correlation between changes in amylase activity and changes in the amount of translatable mRNA as assayed in microinjected Xenopus oocytes. This means that the glucose repression is occurring at a pretranslational stage.

The Interaction of Genetic and Environmental Factors in the Control of Amylase Gene Expression in DROSOPHILA MELANOGASTER.

A number of previous studies have established that amylase activity can vary between Drosophila strains which are maintained under identical laboratory conditions. In addition, we have recently shown that all strains examined so far are subject to glucose repression of amylase activity. In this study, we show that the degree of glucose repression can vary between strains. Moreover, the glucose repression effect is much more pronounced in larvae than in adult flies. Our results lead to the conclusion that the strain-specific differences in activity and the dietary effects are not independent phenomena. These results have implications for the interpretation of many studies on amylase activity variation, including those experiments which have been designed to link amylase activity variations with fitness differences in nature. A question that naturally arises concerns the molecular basis for these strain-specific variations in the degree of glucose repression of this eukaryotic gene.

Prediction of heterosis from DNA fingerprints in chickens.

To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DEP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DEP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DEP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions on SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For "broiler" traits with heterosis of < 1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.

Cloning and expression of a chicken alpha-amylase gene.

We have isolated and sequenced a genomic clone for a pancreatic alpha-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic alpha-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the alpha-amylase produced by the yeast cells was identical to that of the native chicken enzyme.

Cloning and characterization of a bovine genomic fragment homologous to epidermal growth factor genes.

Epidermal growth factor (EGF) is a potent mitogen for a variety of cell types. The 53-amino acid mature EGF protein is encoded by sequences in exons 20 and 21 of a gene spanning over 110 kb. In this study, we report the cloning and characterization of 7.5 kb of bovine genomic sequence homologous to exon 19 through 21 from EGF genes from other mammalian species. The cloned gene fragment had an unusual sequence composition in the form of an in-frame TGA codon in the coding sequence. The sequence was expressed at low levels in kidney tissue and the corresponding cDNA contained the TGA codon. The level of similarity between the bovine exonic sequence and the human, porcine, murine, feline, and canine corresponding sequences varied from 64% to 73%; however, when only sequences encoding the mature EGF protein were compared, the level of similarity between the bovine sequence and the sequence from these species was 59% to 66%. The sequence similarity of the deduced mature protein was lower (34% to 39%) than the sequence similarity of the deduced propeptide. Although the cloned sequences could originate from a bovine EGF pseudogene, the possibility exists that they originate from the functional EGF gene. An as yet unidentified mechanism to by-pass the stop codon would allow the synthesis of a functional EGF protein. Alternatively, the cloned sequence could originate from an EGF-like gene.

Locus-specific diagnostic tests for endogenous avian leukosis-type viral loci in chickens.

The genome of the chicken, Gallus gallus, contains endogenous proviral elements (ALVE elements or ev genes) that display a high degree of similarity to the Avian Leukosis class of retroviruses. The ALVE proviruses are known to modulate physiological processes of the host birds. Different ALVE elements retain variable portions of the complete, prototype viral genome, and each provirus resides in its own specific location within the host genome. Thus, each ALVE element has its own particular potential to modulate host physiology depending on the nature of its integration site, the completeness of the proviral genome, and the level of expression of the locus. It is important, therefore, to be able to establish the ALVE element profiles of chickens quickly and accurately, both in the laboratory and in a commercial setting. The current method of choice for simple, quick, and accurate typing is the polymerase chain reaction (PCR). This paper reviews the present status of PCR typing of ALVE proviruses and lists the assay protocols for 19 different elements. In addition, it compares the insertion sites of these elements in an effort to identify common motifs at ALVE integration sites.

Research note: a rapid method for the detection of the Rous-associated endogenous solitary long terminal repeat, ev15.

A rapid method has been developed for the detection of the solitary long terminal repeat ev15, a member of the avian leukosis virus (ALV) family of endogenous viral elements (ev genes) in chickens. Detection is accomplished by a polymerase chain reaction (PCR) assay that can be performed on purified genomic DNA samples or crude preparations of partially purified whole blood lysates. The test discriminates unambiguously between birds that are homozygous ev15-, homozygous ev15+, or heterozygous ev15-/ev15+. The incorporation of a modified touchdown amplification profile significantly improved the specificity of the PCR assay. Small-scale screening of birds from a variety of chicken breeds has revealed that ev15 is present in populations of both egg-strain birds and broilers.

A new diagnostic method for the detection of endogenous Rous-associated virus-type provirus in chickens.

A quick and simple method has been developed to detect the presence or absence of the endogenous Rous-associated virus (RAV) element ev1 in chickens. The procedure consists of a one-tube multiplex polymerase chain reaction (PCR) involving three oligonucleotide primers that are specific for the upstream flanking region, the long terminal repeat (LTR), and the downstream flanking region of the proviral insert, respectively. The multiplex reaction allows for the unambiguous discrimination between ev1+/ev1+ homozygote, ev1-/ev1- homozygote, and ev1+/ev1- heterozygote birds. The method works best with purified genomic DNA as substrate, but can also be used with rapidly prepared, "crude" DNA samples. The combination of speed with the safety of a nonradioactive procedure, and the ability to perform large numbers of assays by a semi-automated procedure, make this method attractive for large-scale screening projects. The ev1 locus has been used as a model system to demonstrate the feasibility of the PCR diagnostic approach. However the same principle should be applicable to the analysis of other RAV-type ev loci, as well as endogenous elements belonging to other families of viruses as sequence information for the flanking regions of these inserts becomes available.

Screening chickens for endogenous virus ev21 viral element by the polymerase chain reaction.

The molecular architecture of the sex-linked late-feathering region of the chicken genome is still poorly defined. Current evidence points to a strong association between the presence of the endogenous viral element ev21 and the late-feathering phenotype. However, analysis at the molecular level has demonstrated that this is not a simple case of insertional mutagenesis. Instead, the structure of the region of the chicken genome containing the feathering locus is complex and variable between and within lines of chickens. Significant clues to the molecular structure of this genomic region can be obtained by analyzing rare and revertant genotypes. However, searching for rare genotypes can only be carried out effectively using quick screen methodology. This paper describes a quick, polymerase chain reaction-based test for ev21 that facilities the search for rare genotypes.

Characterization of eight endogenous viral (ev) genes of meat chickens in semi-congenic lines.

Restriction fragment length polymorphism analysis was conducted for a set of eight different meat chicken-derived endogenous viral genes (ev genes) of the avian leukosis viral (ALV) family. Each viral element was first isolated into a separate single-element line by selective breeding. Genomic DNA from the founder male for each semi-congenic, single-element line was digested with each of four restriction enzymes, and the resulting Southern blots were each hybridized with up to four probes representing different portions of the ALV retroviral genome. Among the eight elements, there was one that represents the broiler equivalent of locus ev3 of White Leghorn chickens. A second broiler element showed a SacI-specific junction fragment similar to that of ev8. The remainder appeared to be different from any of the 21 ev genes previously described for White Leghorn chickens. Four of the eight elements examined were essentially complete, but the rest have sustained internal deletions.

Characterization of four endogenous viral genes in semi-congenic lines of meat chickens.

Restriction fragment length polymorphism analyses were used to examine endogenous viral genes (ev genes or ALVE genes) of the avian leukosis viral (ALV) family in semi-congenic lines of meat chickens. The Generation 6 lines examined in this study were semi-congenic in that each contained birds with either zero or with one ALVE gene in hemizygous state plus some solitary long terminal repeat (LTR) elements. Using four restriction enzymes on chicken genomic DNA and two probes, one representing the entire ALV retroviral genome and one with only a small part plus the LTR, four ALVE genes were characterized. Each seemed to be complete with no detectable deletions. None appeared to be similar to known ALVE genes of White Leghorns, whereas two of the four may be the same as ALVE genes reported by others in White Plymouth Rock chickens.

Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds.

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M(112)ARQ/T(112)ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr(231)/ARQr(231) and ARQR(231)/ARQr(231) (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens.

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3' portion of the EAV element, extending from the down-stream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20 kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.

A Drosophila gene is subject to glucose repression.

Amylase-specific cDNA probes were used to assay amylase mRNA levels in third-instar larvae of Drosophila melanogaster. It is shown that there is a difference, of the order of 100-fold, in the mRNA levels between larvae that are fed 10% glucose and larvae of the same wild-type strain that are fed an equivalent diet lacking glucose. In fact, the glucose-fed larvae have barely detectable levels of amylase mRNA. This large difference in transcript abundance indicates that the glucose effect, which we previously characterized at the level of enzyme activity, probably reflects a change in the transcriptional activity of the amylase gene.

Long range-inverse PCR (LR-IPCR): extending the useful range of inverse PCR.

The inverse PCR technique (IPCR) has proven to be very useful for the amplification of uncharacterized stretches of DNA upstream or downstream of regions that have already been cloned and sequenced. In practice, however, chromosome walking using standard IPCR is often a slow, repetitive process because only small DNA fragments are effectively amplified. The development of long and accurate PCR methodology has greatly expanded the range of DNA fragment sizes that is amenable to amplification by conventional PCR. We reasoned that combining long range PCR with IPCR would also extend the useful range of the IPCR technique. In this paper we demonstrate the utility of the hybrid, long range-inverse PCR (LR-IPCR) technique by generating clones containing long stretches of DNA flanking endogenous chicken proviral elements.

Molecular biology of enzyme adaptations in higher eukaryotes.

Multicellular eukaryotes have evolved complex homeostatic mechanisms that buffer the majority of their cells from direct interaction with the external environment. Thus, in these organisms long-term adaptations are generally achieved by modulating the developmental profile and tissue specificity of gene expression. Nevertheless, a subset of eukaryotic genes are still involved in direct responses to environmental fluctuations. It is the adaptative responses in the expression of these genes that buffers many other genes from direct environmental effects. Both microevolutionary and macroevolutionary patterns of change in the structure and regulation of such genes are illustrated by the sequences encoding alpha-amylases. The molecular biology and evolution of alpha-amylases in Drosophila and other higher eukaryotes are presented. The amylase system illustrates the effects of both long-term and short-term natural selection, acting on both the structural and regulatory components of a gene--enzyme system. This system offers an opportunity for linking evolutionary genetics to molecular biology, and it allows us to explore the relationship between short-term microevolutionary changes and long-term adaptations.

A general model for the evolution of nuclear pre-mRNA introns.

We present an overview of the evolution of eukaryotic split gene structure and pre-mRNA splicing mechanisms. We have drawn together several seemingly conflicting ideas and we show that they can all be incorporated in a single unified theory of intron evolution. The resulting model is consistent with the notion that introns, as a class, are very ancient, having originated in the "RNA world"; it also supports the concept that introns may have played a crucial role in the construction of many eukaryotic genes and it accommodates the idea that introns are related to mobile insertion elements. Our conclusion is that introns could have a profound effect on the course of eukaryotic gene evolution, but that the origin and maintenance of intron sequences depends, largely, on natural selection acting on the intron sequences themselves.