Molecular insights into Pashmina fiber production: comparative skin transcriptomic analysis of Changthangi goats and sheep.

Ladakh, one of the highest inhabited regions globally, hosts the unique Changthangi goat, renowned for producing Pashmina, the world's most luxurious natural fiber. In comparison, the fiber derived from Changthangi sheep is considered next only to Pashmina. This research endeavors to compare the skin transcriptome profiles of Changthangi goats and Changthangi sheep, aiming to discern the molecular determinants behind the recognition of Changthangi goats as the source of Pashmina. Drawing upon previously conducted studies, a collective of 225 genes correlated with fiber characteristics were extracted from the differentially expressed genes noticed between the two species (p-value of ≤ 0.05 and a log2 fold change of ≥ 1.5). These genes were analyzed using DAVID software to understand their biological functions and to identify enriched KEGG and Reactome pathways. The protein-protein interaction networks were constructed using Cytoscape, cytoHubba, and STRING to focus on key genes and infer their biological significance. Comparative transcriptome analysis revealed significantly higher expression of genes involved in signaling pathways like Wnt, MAPK, PI3K-Akt, Hedgehog, associated with fiber development and quality in Changthangi goats. These pathways play crucial roles in hair follicle (HF) formation, maintenance of epidermal stem cells, and fiber characteristics. Findings also highlight the enrichment of cell adhesion molecules and ECM-receptor interaction, emphasizing their roles in HF structure, growth, and signaling. This investigation offers an in-depth understanding of the molecular intricacies governing Pashmina production in Changthangi goats, providing valuable insights into their unique genetic makeup and underlying mechanisms influencing the exceptional quality of Pashmina fibers.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

BACKGROUND: Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. METHODS: This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. RESULTS: After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. CONCLUSION: The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues.

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Genome-wide characterization and development of SSR markers for genetic diversity analysis in northwestern Himalayas Walnut (Juglans regia L.).

In the present study, we designed and validated genome-wide polymorphic SSR markers (110 SSRs) by mining the walnut genome. A total of 198,924 SSR loci were identified. Among these, successful primers were designed for 162,594 (81.73%) SSR loci. Dinucleotides were the most predominant accounting for 88.40% (175,075) of total SSRs. The SSR frequency was 377.312 SSR/Mb and it showed a decreasing trend from dinucleotide to octanucleotide motifs. We identified 20 highly polymorphic SSR markers and used them to genotype 72 walnut accessions. Over all, we obtained 118 alleles that ranged from 2 to 12 with an average value of 5.9. The higher SSR PIC values indicate their robustness in discriminating walnut genotypes. Heat map, PCA, and population structure categorized 72 walnut genotypes into 2 distinct clusters. The genetic variation within population was higher than among population as inferred by analysis of molecular variance (AMOVA). For walnut improvement, it is necessary to have a large repository of SSRs with high discriminative power. The present study reports 150,000 SSRs, which is the largest SSR repository for this important nut crop. Scientific communities may use this repository for walnut improvement such as QTL mapping, genetic studies, linkage map construction, and marker-assisted selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03563-6.

Potential of carboxymethyl cellulose coating and low dose gamma irradiation to maintain storage quality, inhibit fungal growth and extend shelf-life of cherry fruit.

Carboxymethyl cellulose (CMC) coatings alone and in combination with gamma irradiation was tested for maintaining the storage quality, inhibiting fungal incidence and extending shelf-life of cherry fruit. Two commercial cherry varieties viz. Misri and Double after harvest at commercial maturity were coated with CMC at levels 0.5-1.0 % w/v and gamma irradiated at 1.2 kGy. The treated fruit including control was stored under ambient (temperature 25 ± 2 °C, RH 70 %) and refrigerated (temperature 3 ± 1 °C, RH 80 %) conditions for evaluation of various physico-chemical parameters. Fruits were evaluated after every 3 and 7 days under ambient and refrigerated conditions. CMC coating alone at levels 0.5 and 0.75 % w/v was not found effective with respect to mold growth inhibition under either of the two conditions. Individual treatment of CMC coating at 1.0 % w/v and 1.2 kGy irradiation proved helpful in delaying the onset of mold growth up to 5 and 8 days of ambient storage. During post-refrigerated storage at 25 ± 2 °C, RH 70 %, irradiation alone at 1.2 kGy gave further 4 days extension in shelf-life of cherry varieties following 28 days of refrigeration. All combinatory treatments of CMC coating and irradiation proved beneficial in maintaining the storage quality as well as delaying the decaying of cherry fruit during post-refrigerated storage at 25 ± 2 °C, RH 70 % but, combination of CMC at 1.0 % w/v and 1.2 kGy irradiation was found significantly (p ≤ 0.05) superior to all other treatments in maintaining the storage quality and delaying the decaying of cherry fruit. The above combinatory treatment besides maintaining storage quality resulted in extension of 6 days in shelf life of cherry varieties during post-refrigerated storage at 25 ± 2 °C, RH 80 % following 28 days of refrigeration. Above Combination treatment gave a maximum of 2.3 and 1.5 log reduction in yeast and mold count of cherry fruits after 9 and 28 days of ambient and refrigerated storage, thereby ensuring consumer safety.

Optimizing age of bull at first use in relation to fertility of Murrah breeding bulls.

AIM: The aim of the present investigation was to optimize the age at first use (AAFU) of semen of Murrah breeding bulls, which will help in early selection of bulls under progeny testing program for improving the reproductive performance in the herd. MATERIALS AND METHODS: The data on AAFU, conception rate based on first A.I. (CRFAI), overall conception rate (OCR), and birth weight (B.WT) of 57 Murrah bulls during 1993-2014 at NDRI center pertaining to 14 sets of Network Project on Buffalo Improvement at ICAR-National Dairy Research Institute, Karnal, Haryana, India were adjusted for significant environmental influences and subsequently analyzed. Simple and multiple regression models were used for prediction of CRFAI and OCR of Murrah breeding bulls. Comparative evaluation of three developed models (I-III) showed that Model III, having AAFU and B.WT, fulfill the accuracy of model as revealed by high coefficient of determination, low mean sum of squares due to error, low conceptual predictive value, and low Bayesian information criterion. RESULTS: The results revealed that the average predicted CRFAI was highest (39.95%) at <3.5 years and lowest (34.87%) at >4.5 years of age at first A.I/use. Similarly, average predicted OCR was highest (41.05%) at <3.5 years and lowest (39.42%) at >4.5 years of age at first A.I/use of Murrah bulls. CONCLUSION: In organized herd under progeny testing program, Murrah bulls should be used at young age, i.e. prior to 3.5 years, which is expected to result in 5.08% better CRFAI and 1.63% better OCR in comparison to Murrah bulls used after 4.5 years of age.

Identifying factors affecting age at first semen freezing and age at first semen use in Sahiwal bulls.

AIM: The objective of the study was to evaluate the effects of non-genetic factors on reproduction traits viz. age at first semen freezing and age at first semen use of breeding bulls in Sahiwal bulls by fitting least-squares analysis. MATERIALS AND METHODS: The information on reproduction traits of 43 Sahiwal breeding bulls belonging to 8 sets of Sahiwal breeding program at Indian Council of Agricultural Research-National Dairy Research Institute (ICAR-NDRI), Karnal (Haryana), India during 27 years (1987-2013) were analyzed using fixed linear model. The information was collected from AI records, reproduction sheets, and bull AI register maintained at different sections of Institute viz. record room of Dairy Cattle Breeding Division (DCB), Cattle Yard, Artificial Breeding Research Centre, ICAR-NDRI, Karnal. RESULTS: The average age at first semen freezing and age at first semen use of Sahiwal breeding bulls was estimated as 3.17±0.01 years and 5.35±0.01 years, with the coefficient of variation 18.93% and 20%, respectively. The overall least-squares mean for age at first semen freezing and age at first semen use was estimated as 3.14±0.09 years and 5.25±0.02 years, respectively, in Sahiwal breeding bulls. Period of freezing/use had significant effects on reproductive traits (p<0.01). Season had no significant effect on any of the traits considered in this study. CONCLUSION: It can be concluded that management inputs such as nutrition, breeding, and optimum environment should be taken care of to optimize age at first semen freezing and age at first semen use for better utilization of superior germplasm.

Diarrhoeagenic Escherichia coli and salmonellae in calves and lambs in Kashmir absence, prevalence and antibiogram.

Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India.

AIMS: To study the prevalence and characterize atypical enteropathogenic Escherichia coli (EPEC) and Shiga toxin producing E. coli (STEC) in avian species in India. METHODS AND RESULTS: Two hundred and twelve faecal samples collected from 62 chickens, 50 ducks and 100 pigeons were investigated for the presence of stx(1), stx(2), eae and ehxA virulence genes by multiplex PCR. In all, 42 E. coli isolates (25 chicken, 2 duck and 15 pigeon) possessed at least one virulence gene. Out of these, nine (4.24%) isolates were STEC and 33 (15.56%) were EPEC. All isolates from duck and chicken were EPEC while among 15 pigeon isolates nine (60%) were STEC and six (40%) were EPEC. Among the STEC isolates four each carried stx(1) or stx(2) and one possessed both stx(1) and stx(2). Subtype analysis of stx revealed the presence of stx(2f) in four STEC isolates. None of the STEC isolates carried stx(1c), stx(2c), stx(2d) or stx(2e). Isolates carrying stx(2f) demonstrated vero cell toxicity. One each belonged to serogroup O17 and O78, while one was rough and the other untypeable. All EPEC isolates were atypical as they lacked bfpA. This appears to be the first report of detection of stx(2f) from India. CONCLUSIONS: The study established the presence of stx(1) and stx(2f) containing E. coli in pigeons and atypical EPEC in poultry in India. Pigeons might serve as vectors for transmission of STEC to environment and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons and humans.

Subtype analysis of stx1, stx2 and eae genes in Shiga toxin-producing Escherichia coli (STEC) and typical and atypical enteropathogenic E. coli (EPEC) from lambs in India.

Seventy-five Escherichia coli isolates with at least one targeted virulence gene were recovered from 338 lambs with (n=230) and without (n=108) diarrhoea. The isolates belonged to 36 different serogroups. Shiga toxin-producing E. coli (STEC) was isolated from 9.6% of lambs with and 24.1% of lambs without diarrhoea. Enteropathogenic E. coli (EPEC) was isolated from 6.1% of lambs with and 11.1% of lambs without diarrhoea. Of 26 EPEC isolates, seven were typical (positive for bfpA), and, of 34 stx(1) positive isolates, 25 were subtyped as stx(1c). Five of 29 stx(2) positive isolates were subtyped as stx(2d) and two as stx(2c). Seven of 45 eae positive isolates were subtyped as eae subtype zeta (eaezeta). This appears to be the first report of the isolation of typical EPEC from sheep in India.

Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching.

The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.

Mycophenolate mofetil in the treatment of retroperitoneal fibrosis.

The pathogenesis of idiopathic retroperitoneal fibrosis (IRPF) has been attributed to an autoimmune response to atherosclerotic lipid material leaking from blood vessels. Corticosteroids and cytotoxic agents have been used for therapy. Based on the immunosuppressive and anti-fibrotic action of mycophenolate, we administered this agent to a patient with biopsy-proven IRPF and achieved a rapid, complete and sustained remission with a 6-year follow-up.

Superior vena cava filters: hindsight, insight and foresight.

Upper Extremity Deep Venous Thromboses (UEDVT) are encountered with increasing frequency as the use of central lines and catheters increases. Previously disregarded as benign they are now established to contribute to Pulmonary Embolism (PE) and death. Superior vena cava filters (SVC filters) have been shown to be safe and efficacious for prevention of UEDVT in case anti-coagulation is contraindicated. Compared to Inferior Vena Cava Filters (IVC Filters), data are scarce however and long-term, randomized studies are lacking. SVC filter insertion presents unique indications, contraindications, complications and technical challenges different from their IVC counterparts.

Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle.

A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

Characterization of the RNA chaperone activity of hantavirus nucleocapsid protein.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein, encoded by the smallest of the three genome segments (S), has nonspecific RNA chaperone activity. This activity results in transient dissociation of misfolded RNA structures, may be required for facilitating correct higher-order RNA structure, and may function in viral genome replication. We carried out a series of experiments to further characterize the ability of N to dissociate RNA duplexes. As might be expected, N dissociated RNA duplexes but not DNA duplexes or RNA-DNA heteroduplexes. The RNA-destabilizing activity of N is ATP independent, has a pH optimum of 7.5, and has an Mg(2+) concentration optimum of 1 to 2 mM. N protein is unable to unwind the RNA duplexes that are completely double stranded. However, in the presence of an adjoining single-stranded region, helix unwinding takes place in the 3'-to-5' direction through an unknown mechanism. The N protein trimer specifically recognizes and unwinds the terminal panhandle structure in the viral RNA and remains associated with unwound 5' terminus. We suggest that hantaviral nucleocapsid protein has an active role in hantaviral replication by working cooperatively with viral RNA polymerase. After specific recognition of the panhandle structure by N protein, the unwound 5' terminus likely remains transiently bound to N protein, creating an opportunity for the viral polymerase to initiate transcription at the accessible 3' terminus.

The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3' and 5' ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

Interaction of mithramycin with chromatin.

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.

Association of the anticancer antibiotic chromomycin A(3) with the nucleosome: role of core histone tail domains in the binding process.

The anticancer antibiotic chromomycin A(3) is a transcription inhibitor which forms two types of complexes with Mg(2+): complex I (1:1 in terms of chromomycin A(3)-Mg(2+)) and complex II (2:1 in terms of chromomycin A(3)-Mg(2+)). These complexes are the DNA-binding ligands. With the broad objective of elucidation of the mechanism for action of this group of transcription inhibitors in eukaryotic systems, we have studied the interaction of the antibiotic with nucleosome core particles under different conditions. We have demonstrated and characterized the role of core histone proteins, particularly the N-terminal tail domains, in the association of nucleosome with both complexes of chromomycin. From a scrutiny of the spectroscopic features of the two bound complexes and comparison of the binding and associated thermodynamic parameters, we have shown the following. Core histone(s) stand(s) in the way of access of the ligand(s) to nucleosomal DNA. N-Terminal intact and chopped core particles interact differentially with the same complex. The modes of interaction of the two complexes, I and II, with the same system are different. Tryptic removal of N-terminal tail domains of core histones enhances the binding potential and access of both complexes of chromomycin to the nucleosomal DNA. Agarose gel electrophoresis of an equilibrium mixture containing either complex I or complex II and a saturating concentration of the core particle has demonstrated that both complexes have a tendency to disrupt the nucleosome structure, leading to a release of nucleosomal DNA. Compared to the N-terminal intact nucleosome, the N-terminal chopped nucleosome is more susceptible to disruption. Therefore, we suggest from the above results that the N-terminal tail domains, which have an important role in eukaryotic gene expression, stand in the way of a free access of external agents such as anticancer drugs to the eukaryotic genome. The significance of the results to understand the molecular basis of the transcription inhibitory capacity of chromomycin is discussed.

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+).

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.