Importance of minor groove functional groups for the stability of DNA duplexes

Eight oligonucleotide duplexes have been prepared with four pairs of selected complementary pairs of native/analogue heterocyclic bases incorporated at a selected test site. The base pairs vary in the nature of their functionality in the minor groove. Each pair has a minor groove purine amino group present or absent, and correspondingly has a minor grove pyrimidine carbonyl present or absent. Loss of duplex stability is most notable when the minor groove pyrimidine carbonyl is absent although in other respects normal Watson-Crick hydrogen bonding is maintained in these sequences. These differences in stability are discussed in terms of possible variations in minor groove hydration.     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.     

Germinal centers and the B-cell system

Summary Germinal centers of the rabbit appendix were studied for the presence of complement receptors, immunoglobulin and alkaline phosphatase. In popliteal lymph nodes, de novo-developing germinal centers were studied with respect to these markers up to 21 days after sheep red blood cell (SRBC)-stimulation. In addition, the possible presence of antigen (SRBC) receptor-bearing cells in these germinal centers was investigated.The results may be summarized as follows: 1) Germinal centers in the appendix as well as those in popliteal lymph nodes were rich in complement receptor-bearing cells. Complement-receptor density did not significantly change during a germinal-center reaction. 2) Immunoglobulins were present only at very low densities on the surface of lymphoid cells in the densely populated area of germinal centers. In germinal centers of popliteal lymph nodes lymphoid cells in the thinly populated area again showed higher densities. Immunoglobulins possibly complexed with antigen on the surface of follicular dendritic cells were not observed in the initial phase of a germinal center reaction. In contrast, in germinal centers of the appendix, immunoglobulin was present in excessive amounts throughout the thinly populated area, possibly complexed with antigen, which is also abundantly present. 3) Reticular staining patterns of alkaline phosphatase were present in the densely populated area, but absent in the thinly populated area of germinal centers in both appendix and popliteal lymph nodes. Primary follicles and young germinal centers were alkaline phosphatase-negative. 4) Antigen receptor-bearing cells were detected in germinal centers of popliteal lymph nodes as early as 5 days after SRBC-stimulation, reaching a maximum at day 10.In conclusion, with the present experimental approach, microenvironmental differences were shown between the densely populated area and the thinly populated area of germinal centers. However, no indication was obtained for a postulated maturation event of the lymphocytes within germinal centers, or for functional differences that may exist between germinal centers in the appendix and popliteal lymph nodes.     

Diversity of mammary tumor viral genes within the genus Mus, the species Mus musculus, and the strain C3H.

Proviral sequences complementary to the C3H mouse mammary tumor virus RNA genome are present in the DNA of early occurring mammary tumors of C3H/HeN mice and are absent from apparently normal C3H/HeN tissues; these sequences are non-germ line transmitted in C3H/HeN mice and have been termed tumor-associated sequences; (W. Drohan et al., J. Virol. 21:986-995, 1977). We report here that tumor-associated sequences are present in the DNA of spontaneous mammary tumors that occur early in the life of several inbred, high-tumor-incidence mouse strains but are absent in mammary tumors that occur later in life in low- and moderate-tumor-incidence strains. These sequences are also absent in apparently normal organs tested from numerous laboratory mouse strains, feral mice, Mus musculus subspecies, and other Mus species. Sequences represented in tumor-associated sequence RNA, however, are present as endogenous provirus in GR mice (at approximately four copies per haploid genome) and in two of five substrains of C3H mice tested (at approximately one copy per haploid genome). The two substrains of C3H mice positive for endogenous tumor-associated sequence provirus were recently (circa 1930) separated from the negative substrains of C3H mice. The results may be explained by the unlikely chance segregation of proviral sequences or by the recent integration of viral genes (within the last few decades). Whereas radioactively labeled mouse mammary tumor virus 60-70S RNA or complementary DNA detected mouse mammary tumor virus-related proviral information in all laboratory mouse strains, feral mice, subspecies of M. musculus, and other species of Mus, the use of tumor-associated sequence RNA clearly revealed the genetic diversity that may exist between different colonies or substrains of "inbred" laboratory mice commonly used in cancer research.     

The Evolution of Acetyl-CoA Synthase

Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoA from CO2 + 2e-, CoA, and a methyl group, and contain 5 types of subunits (alpha, beta, gamma, delta, and epsilon). Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition. Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an alpha 2 beta 2 tetramer and a gamma delta heterodimer. Class IV enzymes catabolize CO to CO2 and are alpha-subunit monomers. Phylogenetic analyses were performed on all five subunits. ACS alpha sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residues that may function as ligands to the B- and C-clusters were identified. Other residues exclusively conserved in Class I/II sequences may be ligands to additional metal centers in Class I and II enzymes. ACS beta sequences also separated into two groups, but they were less divergent than the alpha's, and the separation was not as distinct. Class III-like beta sequences contained approximately 300 residues at their N-termini absent in Class I/II sequences. Conserved residues identified in beta sequences may function as ligands to active site residues used for acetyl-CoA synthesis. ACS gamma-sequences separated into 3 groups (Classes I, II, and III), while delta-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of alpha sequences. ACS epsilon-sequence topology showed greater divergence and less consistency vis-à-vis the other subunits, possibly reflecting reduced evolutionary constraints due to the absence of metal centers. The alpha subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms may have evolved are discussed.     

A DNA fragment from the human X chromosome short arm which detects a partially homologous sequence on the Y chromosomes long arm.

An X linked human DNA fragment (named DXS31 ) which detects partially homologous sequences on the Y chromosome has been isolated. Regional localisation of the two sex linked sequences was determined using a panel of rodent-human somatic cell hybrids. The X specific sequence is located at the tip of the short arm ( Xp22 .3-pter), i.e. within or close to the region which pairs with the Y chromosome short arm at meiosis. However the Y specific sequence is located in the heterochromatic region of the long arm ( Yq11 -qter) and lies outside from the pairing region. DNAs from several XX male subjects were probed with DXS31 and in all cases a double dose of the X linked fragment was found, and the Y specific fragment was absent. DXS31 detects in chimpanzee a male-female differential pattern identical to that found in man. However results obtained in a more distantly related species, the brown lemur, suggest that the sequences detected by DXS31 in this species might be autosomally coded. The features observed with these X-Y related sequences do not fit with that expected from current hypotheses of homology between the pairing regions of the two sex chromosomes, nor with the pattern observed with other X-Y homologous sequences recently characterized. Our results suggest also that the rule of conservation of X linkage in mammals might not apply to sequences present on the tip of the X chromosome short arm, in bearing with the controversial issue of steroid sulfatase localisation in mouse.     

Differential subtraction chain, a method for identifying differences in genomic DNA and mRNA.

Identifying the genetic differences between two organisms or cell types has been a major goal in modern biomedical research. Recently, we developed a novel methodology that can rapidly identify the differences between two populations of DNA. This method, termed 'differential subtraction chain' (DSC), is based on a novel 'negative amplification' strategy that converts (amplifiable) tester sequences to counterpart (unamplifiable) driver sequences. The result is a double exponential elimination of amplifiable sequences in the testers, while preserving the sequences in the testers that have no counterpart in the drivers. We applied this methodology to the genome of a glioblastoma cell line. A homozygous deletion was rapidly identified. We extended this technique to identifying the unique sequences in mRNA. Two CDC25 transgene fragments were quickly identified in a cdc25B transgenic mouse. We also applied this methodology to systems with profound differences in mRNA expression. In a 'prostate epithelia subtracting blood cells' DSC reaction, a sample of unique gene fragments which are absent in the prostate but present in the blood were identified. Lastly, we detected rare (1 virus/100 cells) Herpes simplex virus type 2 (HSV-2) sequences in a tissue culture, indicating good sensitivity of this methodology. Overall, DSC represents a fast, efficient and sensitive method for identifying differences in genomic DNA and mRNA and can be easily applied in a variety of biological systems.     

Chromosomal distribution of repetitive DNA sequences highlights the independent differentiation of multiple sex chromosomes in two closely related fish species

The arrangement of 6 repetitive DNA sequences in the mitotic and meiotic sex chromosomes of 2 Erythrinidae fish, namely Hoplias malabaricus and Erythrinus erythrinus, both with a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y sex chromosome system, was analyzed using fluorescence in situ hybridization. The distribution patterns of the repetitive sequences were distinct for each species. While some DNA repeats were species-specific, others were present in the sex chromosomes of both species at different locations. These data, together with the different morphological types of sex chromosomes and the distinct chromosomal rearrangements associated with the formation of the neo-Y chromosomes, support the plasticity of sex chromosome differentiation in the Erythrinidae family. Our present data highlight that the sex chromosomes in fish species may follow diverse differentiation patterns, even in the same type of sex chromosome system present in cofamiliar species.     

Are human natal sex ratio differences across the world adaptive? A test of Fisher's principle

Fisher''s principle states that natural selection favours an equal number of male and female births at the population level, unless there are sex differences in rearing costs or sex differences in mortality before the end of the period of parental investment. Sex differences in rearing costs should be more pronounced in low- than in high-resource settings. We, therefore, examined whether human development index and sex differences in child mortality contribute to the natural variation in human sex ratio at birth across the globe. As predicted by Fisher''s principle, the proportion of male births increased with both increasing male-biased childhood mortality and level of development of each country. However, these relationships were absent after accounting for spatial autocorrelation in the residuals, which our inference is conditioned on. This work shows how the failure to account for residual spatial autocorrelation can lead to incorrect conclusions regarding support for predictions from sex allocation theory.     

Sex differences in the vocal repertoire of adult red‐capped mangabeys (Cercocebus torquatus): a multi‐level acoustic analysis

Sex differences in the vocal behavior of nonhuman primates can take various forms: sex‐specific call types, differential production of shared call types, or sex discrepancy in phonation. Also, a growing literature is evidencing that systematically analyzing the vocal repertoires of primates at the call level might lead to underestimating their communicative abilities. Here, we present an extensive multi‐level analysis of the still unknown vocal repertoire of adult red‐capped mangabeys (Cercocebus torquatus), with a special emphasis on sex differences. We collected recordings from seven adult males and seven adult females housed in captivity. We present a structurally‐based classification of mangabey calls that we cross‐validated by an analysis of the associated contexts of emission. We found 12 sound units (including six sex‐specific) that were concatenated to form eight call types (including four sex‐specific), which were produced either singularly or in sequences composed of one (“repetition”) or several (“combination”) call types. We extracted organizational principles that ruled call composition and calling patterns. This revealed a high degree of potentially meaningful variability in terms of semantics and syntax. Male–female discrepancy in terms of phonation could be related to morphological dimorphism and would enable listeners to behave appropriately according to the sex of the caller. Sex differences in repertoire size, structural gradation, and call usage could reflect specificities of male–female social roles. We discuss the pertinence of these sex differences according to social system and habitat quality. Am. J. Primatol. 72:360–375, 2010. © 2010 Wiley‐Liss, Inc.     

Development of an X-specific marker and identification of YY individuals in spinach

Spinach is a popular vegetable native to central and western Asia. It is dioecious with a pair of nascent sex chromosomes. The difficulties of working with the non-recombining sex determination region of XY individuals have hindered the progress toward sequencing sex chromosomes of most dioecious species. Here we present important advances toward characterizing the non-recombining sex chromosomes in spinach. Of nearly 400 spinach accessions screened, we identified a single accession of spinach in which androdioecious XY individuals segregate YY spinach. The male and female genomes of the spinach cultivar Shami and USDA accession PI 664497 were sequenced at 12–17?× coverage. X-specific sequences were identified by comparing the depth of coverage differences between male and female alignments to a female draft genome. YY individuals were used as a negative control to validate X-specific markers found by depth of coverage analysis. Of 19 possible X chromosome sequences found by depth of coverage analysis, one was verified to be X-specific by a PCR-based marker, SpoX, which amplified genomic DNA from XX and XY, but not YY templates. Androdioecious XY individuals of accession PI 217425 (Cornell #9) were used to develop inbred lines, and at S7 generation, all XY individuals were androdioecious and all YY individuals were pure male. The sex reversal of the XY mutant to hermaphrodite is strong evidence that the sex chromosomes in spinach have a two-gene sex determination system. These results are crucial towards sequencing the X and Y chromosomes to advance sex chromosome research in spinach.     

Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Sry-negative XX sex reversal in the American cocker spaniel dog

The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley-Liss, Inc.     

The use of (GACA)4 PCR to sex Old World vultures (Aves: Accipitridae)

A PCR method was developed employing a single primer (GACA)₄ to sex Black vultures ( Aegypius monachus ), Lappet-faced vultures ( Torgos tracheliotus ), and Griffon vultures ( Gyps fulvus ). Using the (GACA)₄ primer several PCR products were generated. One or more PCR products displayed a sex-specific pattern, i.e. they were only present in females (probably corresponding to repetitive DNA on the W chromosome) but absent in males. The sex ratio of 85 Griffon vultures from Spain was almost 1.     

由不同细胞核背景引起的小麦细胞质雄性不育系(CMS)叶绿体蛋白质组的变化(简报)

叶绿体和线粒体是高等植物细胞内2种重要的细胞器。由于细胞质雄性不育(CMS)被认为是一种由线粒体基因编码的性状,因此,近10多年来,国内外研究者对线粒体基因组结构与功能、由线粒体基因编码的与CMS相关蛋白的研究积累了大量的资料。与线粒体相比,叶绿体与CMS关系的研究相对滞后。虽然一些研究者在核不育水稻中,观     

The microscopic anatomy of the human pharyngeal tonsil in the postnatal period of ontogeny

Histological sections of the pharyngeal tonsil (PhT) have been investigated in 55 corpses of persons of both sex and various age, that had no disease connected with lesions of the PhT by the time of their death. The aim of the work is to determine relative areas of each structural element of the PhT in comparison to the whole section area taken as 100%. The PhT lymphoid tissue reached its maximal relative development at the first childhood. Then the lymphoid tissue area in the PhT histological sections decreases gradually, making already in the second mature age one half of the tonsil section area, and in the elderly age--nearly one-fourth. The decrease of the relative lymphoid tissue area takes place at the expense of decreasing area of the lymphoid nodules. The relative area of the connective stroma increases inversely to the relative decrease of the lymphoid tissue area parameters. The germinative centers in the lymphoid nodules are absent in newborns and old persons. The greatest value of the relative area of the germinative centers falls to the early infancy. The lymphoid nodules with the germinative centers, according to their relative area during all age periods, besides the newborn period, the second mature, elderly and old age periods, essentially exceed the area, that occupy the nodules without the germinative centers on the PhT sections.