Transcriptome analysis of blastoderms exposed to prolonged egg storage and short periods of incubation during egg storage.

BACKGROUND: Cool temperature egg storage prior to incubation is a common practice in the broiler industry; however, prolonged egg storage causes increased embryonic mortality and decreased hatchability and growth in surviving chicks. Exposing eggs to short periods of incubation during egg storage (SPIDES) reduces the adverse consequences of prolonged storage. SPIDES increases blastodermal cell viability by reducing apoptosis, though the counteracting mechanisms are unclear. To define the impact of prolonged storage and SPIDES, transcriptome analysis compared gene expression from blastoderms isolated from eggs exposed to the following treatments: control (CR, stored at 17 °C for 4 days), prolonged storage (NSR, stored at 17 °C for 21 days), SPIDES (SR, stored at 17 °C for 21 days with SPIDES), and incubated control (C2, stored at 17 °C for 4 days followed by incubation to HH (Hamburger-Hamilton) stage 2, used as the ideal standard development) (n = 3/group). Data analysis was performed using the CLC Genomics Workbench platform. Functional annotation was performed using DAVID and QIAGEN Ingenuity Pathway Analysis. RESULTS: In total, 4726 DEGs (differentially expressed genes) were identified across all experimental group comparisons (q < 0.05, FPKM> 20, |fold change| > 1.5). DEGs common across experimental comparisons were involved in cellular homeostasis and cytoskeletal protein binding. The NSR group exhibited activation of ubiquitination, apoptotic, and cell senescence processes. The SR group showed activation of cell viability, division, and metabolic processes. Through comparison analysis, cellular respiration, tRNA charging, cell cycle control, and HMBG1 signaling pathways were significantly impacted by treatment and potential regulatory roles for ribosomal protein L23a (RPL23A) and MYC proto-oncogene, BHLH transcription factor (MYC) were identified. CONCLUSIONS: Prolonged egg storage (NSR) resulted in enriched cell stress and death pathways; while SPIDES (SR) resulted in enriched basic cell and anti-apoptotic pathways. New insights into DNA repair mechanisms, RNA processing, shifts in metabolism, and chromatin dynamics in relation to egg storage treatment were obtained through this study. Although egg storage protocols have been examined through targeted gene expression approaches, this study provided a global view of the extensive molecular networks affected by prolonged storage and SPIDES and helped to identify potential upstream regulators for future experiments to optimize egg storage parameters.

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway.

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Central mutation databases--a review.

The Internet has been a key component in the coordination of the diverse group of scientists involved in the Human Genome Project. Nowhere has this contribution been more critical than in the maintenance and exchange of information about genetic variation and mutation. Whereas the majority of DNA sequence is generated and stored by a relatively few sites, a far greater number of researchers investigate the variations in that sequence from sites scattered worldwide. It falls to central databases to utilize the Internet to assemble data from these sites and make them available to the greater human genomic community.

The tetranucleotide repeat polymorphism D21S1245 demonstrates hypermutability in germline and somatic cells.

Six novel polymorphic short sequence repeats were identified and localized on the linkage map of human chromosome 21 by genotyping the CEPH reference pedigrees. One of these markers, the tetrameric (AAAG)n repeat D21S1245, was found to be hypermutable. In the DNAs from lymphoblastoid cell lines of members of the 40 CEPH families a total of 18 new alleles were detected. These new alleles, sometimes appearing in mosaic forms, arose equally in paternal and maternal DNAs, and could be equally larger or smaller than the alleles from which they were derived. The larger alleles of D21S1245 are more prone to be converted to new alleles. None of the new alleles with mosaicism were present in the corresponding genomic blood DNA, and therefore originated during or after the establishment of the lymphoblastoid cell lines; half of the new alleles without mosaicism were also found in genomic blood DNA of the appropriate CEPH individuals. The range of germline mutation rate observed in the 716 meioses examined was 0.56-1.4 x 10(-2); the range of somatic mutations observed in the 405 cell lines examined was 1.96-3.46 x 10(-2). This is one of the most hypermutable microsatellite repeat polymorphism in the human genome detected to date. D21S1245, is highly polymorphic (heterozygosity of 0.96) and maps between D21S231 and D21S198.

Two craniosynostotic syndrome loci, Crouzon and Jackson-Weiss, map to chromosome 10q23-q26.

Crouzon syndrome (MIM 123500) is a common autosomal dominant form of craniosynostosis with shallow orbits, ocular proptosis, and maxillary hypoplasia. Jackson-Weiss syndrome (MIM 123150) is another autosomal dominant craniosynostosis with highly variable phenotypic expression. Unlike Crouzon syndrome, Jackson-Weiss syndrome is associated with foot anomalies. We performed two point linkage and haplotype analyses using 13 dinucleotide repeat markers on chromosome 10, spanning a genetic distance of 108 cM. The Crouzon syndrome locus (CFD1) maps to the region of chromosome 10q2, with the tightest linkage to locus D10S205 (Z = 3.09, theta = 0.00). the Jackson-Weiss syndrome locus in the large Amish pedigree in which the condition was originally described was also linked to the chromosome 10q23-q26 region between loci D10S190 and D10S186. The D10S209 locus was most strongly linked (Z = 11.29, theta = 0.00).

Maternal uniparental disomy for human chromosome 14, due to loss of a chromosome 14 from somatic cells with t(13;14) trisomy 14.

Uniparental disomy (UPD) for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans. We recently studied a 9-year-old female with a de novo Robertsonian translocation t(13;14), short stature, mild developmental delay, scoliosis, hyperextensible joints, hydrocephalus that resolved spontaneously during the first year of life, and hypercholesterolemia. To determine the parental origin of chromosomes 13 and 14 in the proband, we have studied the genotypes of DNA polymorphic markers due to (GT)n repeats in the patient and her parents' blood DNA. The genotypes of markers D14S43, D14S45, D14S49, and D14S54 indicated maternal UPD for chromosome 14. There was isodisomy for proximal markers and heterodisomy for distal markers, suggesting a recombination event on maternal chromosomes 14. In addition, DNA analysis first revealed--and subsequent cytogenetic analysis confirmed--that there was mosaic trisomy 14 in 5% of blood lymphocytes. There was normal (biparental) inheritance for chromosome 13, and there was no evidence of false paternity in genotypes of 11 highly polymorphic markers on human chromosome 21. Two cases of maternal UPD for chromosome 14 have previously been reported, one with a familial rob t(13;14) and the other with a t(14;14). There are several similarities among these patients, and a "maternal UPD chromosome 14 syndrome" is emerging; however, the contribution of the mosaic trisomy 14 to the phenotype cannot be evaluated. The study of de novo Robertsonian translocations of the type reported here should reveal both the extent of UPD in these events and the contribution of particular chromosomes involved in certain phenotypes.

Mitotic errors in somatic cells cause trisomy 21 in about 4.5% of cases and are not associated with advanced maternal age.

The study of DNA polymorphisms has permitted the determination of the parental and meiotic origin of the supernumerary chromosome 21 in families with free trisomy 21. Chromosomal segregation errors in somatic cells during mitosis were recognized after analysis of DNA markers in the pericentromeric region and (in order to identify recombination events) along the long arm of chromosome 21. Mitotic errors accounted for about 4.5% (11 of 238) of free trisomy 21 cases examined. The mean maternal age of mitotic errors was 28.5 years and there was no association with advanced maternal age. There was no preference in the parental origin of the duplicated chromosome 21. The 43 maternal meiosis II errors in this study had a mean maternal age of 34.1 years-the highest mean maternal age of all categories of chromosomal segregation errors.

DNA polymorphism analysis in families with recurrence of free trisomy 21.

We used DNA polymorphic markers on the long arm of human chromosome 21 in order to determine the parental and meiotic origin of the extra chromosome 21 in families with recurrent free trisomy 21. A total of 22 families were studied, 13 in which the individuals with trisomy 21 were siblings (category 1), four families in which the individuals with trisomy 21 were second-degree relatives (category 2), and five families in which the individuals with trisomy 21 were third-degree relatives, that is, their parents were siblings (category 3). In five category 1 families, parental mosaicism was detected, while in the remaining eight families, the origin of nondisjunction was maternal. In two of the four families of category 2 the nondisjunctions originated in individuals who were related. In only one of five category 3 families, the nondisjunctions originated in related individuals. These results suggest that parental mosaicism is an important etiologic factor in recurrent free trisomy 21 (5 of 22 families) and that chance alone can explain the recurrent trisomy 21 in many of the remaining families (14 of 22 families). However, in a small number of families (3 of 22), a familial predisposing factor or undetected mosaicism cannot be excluded.

The human serum amyloid A locus SAA4 is a pseudogene.

We have isolated the human genomic DNA clone GSAA4 from a size-selected Bgl II library by hybridization to a probe derived from the human serum amyloid A gene GSAA1. Sequencing the 5' end of this clone revealed a region similar to the first exon of gene GSAA1 but with significant nucleotide differences and mutation of the 3' splice site. The restriction map of the GSAA4 clone corresponds to that for the locus "SAA4" recently reported by others. Sequence and hybridization details indicate that the locus in clone GSAA4 is a member of the human serum amyloid A gene family and contains a pseudogene. Isolating GSAA4 completes the collection of clones needed to account for all bands found in blot hybridizations of human DNA using serum amyloid A gene probes.

Exclusion of linkage between familial Mediterranean fever and the human serum amyloid A (SAA) gene cluster.

We studied the relationship between the autosomal recessive trait familial Mediterranean fever (FMF) and the serum amyloid A (SAA) genes by comparing alleles of a highly polymorphic dinucleotide repeat and a conventional restriction fragment length polymorphism (RFLP) in the SAA gene cluster in Israeli FMF kindreds. By haplotype analysis, our data indicate a minimum crossover frequency of 22% between the SAA gene marker and FMF. By conventional linkage analysis this eliminates a minimum of 10.4 cM including and surrounding the SAA gene cluster as the site of the FMF mutation although SAA proteins are prominent physiologic markers of the acute attacks.

Highly polymorphic domains of the human serum amyloid A (SAA) gene GSAA1.

The DNA of the human serum amyloid A (SAA) gene GSAA1 contains several repetitive regions within its introns. We have studied length variations at one such region in the 2nd intron by selective amplification using the polymerase chain reaction (PCR) and defined oligonucleotide primers. The lengths of the repetitive regions frequently differ between individual chromosomes and can be transmitted as Mendelian markers, making them useful for genetic linkage analysis.

The human serum amyloid A (SAA)-encoding gene GSAA1: nucleotide sequence and possible autocrine-collagenase-inducer function.

We have determined the genomic sequence of the human GSAA1 gene, a member of the family of acute-phase human serum amyloid A (SAA)-encoding genes. This sequence predicts a mature protein of 104 amino acids (aa), several of which differ from residues usually conserved in the sequence of SAA proteins isolated from serum. Despite coding differences, however, the four-exon structure of GSAA1 resembles that of other SAA genes in humans and mice. The N-terminal 25 aa of the mature GSAA1 protein are virtually identical to those of an 'SAA-like' autocrine collagenase inducer produced by rabbit synovial fibroblasts; the latter also differ from the corresponding aa found in SAA in serum. We propose that GSAA1 is the human gene coding for a protein closely related to the SAA, but which is adapted to this important autocrine cytokine function.

Molecular analysis of the human serum amyloid A (SAA) gene family.

We have assigned the human serum amyloid A (SAA) gene family to a 90 kb region on the short arm of human chromosome 11 (11p) by hybridization of defined genomic fragments of human SAA genes to DNA from rodent-human somatic cell hybrids and to large DNA fragments separated by transverse alternating field gel electrophoresis. We have also characterized SAA probe hybridization patterns in human DNA cleaved with restriction endonucleases Hind III, Pst I, BglII, TaqI, and XbaI and found invariant patterns except for a two-allele restriction fragment length polymorphism (RFLP) with Hind III. These studies show that the SAA gene family comprises at least three members in the haploid human genome and will be useful in identifying variant patterns and establishing linkage between members of the SAA gene family and other markers on chromosome 11.

Repeated DNA of the human Y chromosome.

A significant fraction of the human Y chromosome is composed of DNA sequences which have homologues on the X chromosome or autosomes in humans and non-human primates. However, most human Y-chromosome sequences so far examined do not have homologues on the Y chromosomes of other primates. This observation suggests that a significant proportion of the human Y chromosome is composed of sequences that have acquired their Y-chromosome association since humans diverged from other primates. More than 50% of the human Y chromosome is composed of a variety of repeated DNAs which, with one known exception, can be distinguished from homologues elsewhere in the genome. These include the alphoid repeats, the major human SINE (Alu repeats) and several additional families of repeats which account for the majority of Y-chromosome repeated DNA. The alphoid sequences tandemly clustered near the centromere on the Y chromosome can be distinguished from those on other chromosomes by both sequence and repeat organization, while the majority of Y-chromosome Alu repeats have little homology with genomic consensus Alu sequences. In contrast, the Y-chromosome LINE repeats cannot be distinguished from LINEs found on other chromosomes. It has been proposed that both SINE and LINE repeats have been dispersed throughout the genome by mechanisms that involve RNA intermediates. The difference in the relationship of the Y-chromosome Alu and LINE repeats to their respective family members elsewhere in the genome makes it possible that their dispersal to the Y chromosome has occurred by different mechanisms or at different rates. In addition to the SINE and LINE repeats, the human Y chromosome contains a group of repeated DNA elements originally identified as 3.4 and 2.1 kb fragments in HaeIII digests of male genomic DNA. Although the 3.4 and 2.1 kb Y repeats do not cross-react, both exist as tandem clusters of alternating Y-specific and non-Y-specific sequences. The 3.4 kb Y repeats contain at least three distinct sequences with autosomal homologies interspersed in various ways with a collection of several different Y-specific repeat sequences. Individual recombinant clones derived from isolated 3.4 kb HaeIII Y fragments have been identified which do not cross-react. Thus, the 3.4 kb HaeIII Y fragments are a heterogeneous mixture of sequences which have in common the regular occurrence of HaeIII restriction sites at 3.4 kb intervals and an organization as tandem clusters at various sites along the Y-long arm.(ABSTRACT TRUNCATED AT 400 WORDS)

Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex.

Increased production of fetal hemoglobin (HbF) was observed in a patient with sickle cell anemia treated with 5-azacytidine. Each of four courses of therapy resulted in a rapid and prolonged increase in the percentage of HbF containing reticulocytes (F reticulocytes) and HbF containing erythrocytes (F cells). The percentage of HbF in peripheral blood rose from 1.8 to 8.9%. The rise in HbF production was accompanied by an increase in peripheral blood hemoglobin concentration from 8 to 12 g/dl and an increase in mean erythrocyte volume. Treatment with 5-azacytidine resulted in hypomethylation of total genomic and a Y-chromosome-specific DNA fragment isolated from both peripheral blood and bone marrow. Of 15 restriction enzyme sites around the gamma-delta-beta-globin gene complex, only 2 became hypomethylated: one 107 bases 5' to the gamma G and the other 107 bases 5' to the gamma A globin genes.

5-Azacytidine increases fetal hemoglobin production in a patient with sickle cell disease.

5-Azacytidine, given to one sickle cell patient as 9 doses of 30 mg/m2 each at intervals of 8 hr, rapidly increased the number of red cells containing HbF (F cells), decreased the amount of HbS in F cells, and decreased the MCHC of F cells. Although a significant rise in the peripheral blood hemoglobin from 8 gm/dl to 10-12 gm/dl was obtained, painful vaso-occlusive crises continued to occur. Therapy was associated with no marrow toxicity, although erythroblastosis appeared after each course of therapy. Increased HbF production was associated with demethylation of two restriction enzyme sites 5' to the two gamma-globin genes. Five other restriction sites around the gamma-globin genes were unchanged. 5-Azacytidine is considered potentially mutagenic and carcinogenic in man and, therefore, further experimental treatment should be limited only to severely affected sickle cell patients.

Population heterogeneity of the Hpa I restriction site associated with the beta globin gene: implications for prenatal diagnosis.

The Hpa I restriction endonuclease site polymorphism that results in some human beta globin genes being contained in a 13-kilobase (kb) DNA restriction fragment rather than in the usual 7.6-kb fragment has been reported to be in linkage disequilibrium with the beta S mutation. The frequency of the 13-kb fragment among Baltimore black sickle cell (SS) disease patients (58%) is lower than that reported for San Francisco black SS disease patients (87%) and similar to that reported for such New York patients (59%). There is, then, considerable heterogeneity among American black populations. Therefore, for the purposes of prenatal diagnosis, the frequency in the particular population at risk should be established. When the frequency of association of the 13-kb fragment and the beta S mutation is low, the linkage phase must also be established. When the linkage phase is known, the Hpa I pattern alone can exclude SS disease 54% of the time for Baltimore AS X AS couples.

Partial purification and characterization of DNA from the human X chromosome.

Human X chromosome DNA was partially purified from a mouse-human hybrid cell line containing a single human chromosome, the X. Enrichment of such DNA was accomplished by two sequential reassociations of radiolabeled hybrid cell DNA with large excesses of mouse DNA. Unreassociated hybrid cell DNA was used as a probe for human X chromosome sequences. The human-specific fraction of probe DNA CONTAINED THREE COMPONENTS. Two of these reassociated to human DNAs at rates proportional to the number of X chromosomes present. These two components were thus localized to the X chromosome. One of these X-specific components, representing about 80% of human-specific probe DNA, consisted of single copy or very low order reiterated DNA. The second X-specific component, representing about 10% of human-specific probe DNA, was about 20-30 times more reiterated. The remaining 10% of human-specific probe DNA, although derived from the X chromosome, reassociated to human DNAs at a rate independent of the number of X chromosomes present. This component was thus homologous to autosomal as well as X chromosome DNA. The probe DNA accounts for approximately half of the human X chromosome, suggesting that the remainder may have homology with mouse DNA.