A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

Fragments of ATM which have dominant-negative or complementing activity.

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.     

Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is the most common microdeletion syndrome in humans. It occurs with an estimated frequency of 1 in 4, 000 live births. Most cases occur sporadically, indicating that the deletion is recurrent in the population. More than 90% of patients with VCFS and a 22q11 deletion have a similar 3-Mb hemizygous deletion, suggesting that sequences at the breakpoints confer susceptibility to rearrangements. To define the region containing the chromosome breakpoints, we constructed an 8-kb-resolution physical map. We identified a low-copy repeat in the vicinity of both breakpoints. A set of genetic markers were integrated into the physical map to determine whether the deletions occur within the repeat. Haplotype analysis with genetic markers that flank the repeats showed that most patients with VCFS had deletion breakpoints in the repeat. Within the repeat is a 200-kb duplication of sequences, including a tandem repeat of genes/pseudogenes, surrounding the breakpoints. The genes in the repeat are GGT, BCRL, V7-rel, POM121-like, and GGT-rel. Physical mapping and genomic fingerprint analysis showed that the repeats are virtually identical in the 200-kb region, suggesting that the deletion is mediated by homologous recombination. Examination of two three-generation families showed that meiotic intrachromosomal recombination mediated the deletion.     

Der(22) syndrome and velo-cardio-facial syndrome/DiGeorge syndrome share a 1.5-Mb region of overlap on chromosome 22q11

Derivative 22 (der[22]) syndrome is a rare disorder associated with multiple congenital anomalies, including profound mental retardation, preauricular skin tags or pits, and conotruncal heart defects. It can occur in offspring of carriers of the constitutional t(11;22)(q23;q11) translocation, owing to a 3:1 meiotic malsegregation event resulting in partial trisomy of chromosomes 11 and 22. The trisomic region on chromosome 22 overlaps the region hemizygously deleted in another congenital anomaly disorder, velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Most patients with VCFS/DGS have a similar 3-Mb deletion, whereas some have a nested distal deletion endpoint resulting in a 1.5-Mb deletion, and a few rare patients have unique deletions. To define the interval on 22q11 containing the t(11;22) breakpoint, haplotype analysis and FISH mapping were performed for five patients with der(22) syndrome. Analysis of all the patients was consistent with 3:1 meiotic malsegregation in the t(11;22) carrier parent. FISH-mapping studies showed that the t(11;22) breakpoint occurred in the same interval as the 1.5-Mb distal deletion breakpoint for VCFS. The deletion breakpoint of one VCFS patient with an unbalanced t(18;22) translocation also occurred in the same region. Hamster-human somatic hybrid cell lines from a patient with der(22) syndrome and a patient with VCFS showed that the breakpoints occurred in an interval containing low-copy repeats, distal to RANBP1 and proximal to ZNF74. The presence of low-copy repetitive sequences may confer susceptibility to chromosome rearrangements. A 1.5-Mb region of overlap on 22q11 in both syndromes suggests the presence of dosage-dependent genes in this interval.     

Alleviation of water and osmotic stress-induced changes in nitrogen metabolizing enzymes in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars by potassium

Present communication reports laboratory and pot experiments conducted to study the influence of water and osmotic stress on nitrogen uptake and metabolism in two wheat (Triticum aestivum L) cultivars with and without potassium supplementation. Polyethylene glycol 6000-induced osmotic stress/restricted irrigation caused a considerable decline in the activity of nitrate reductase, glutamate synthase, alanine and aspartate aminotransferases, and glutamate dehydrogenase. Potassium considerably improved nitrogen metabolism under normal water supply conditions and also resulted in amelioration of the negative impact of water and osmotic stresses indicating that potassium supplementation can be used as a potential tool for enhancing the nitrogen use efficiency in wheat for exploiting its genetic potential.     

Age-related and seasonal variations in plasma uncarboxylated osteocalcin in male Murrah buffaloes

The present study aims to investigate the age-related and seasonal variations in plasma uncarboxylated osteocalcin (UcOCN) in male Murrah buffaloes. In experiment 1, body weight, body dimensions and plasma UcOCN levels were estimated in 120 male Murrah buffaloes aged between 18 days and 8 years. We observed a significant (p < 0.0001) decrease in plasma UcOCN with age and their levels ranged between 0.25 and 9.25 ng/ml. In experiment 2, the plasma UcOCN, cortisol, triiodothyronine (T₃), thyroxine (T₄) and prolactin levels were determined during spring and summer in 6–34 months (n = 24) old male Murrah buffaloes. Significant (p < 0.0001) higher plasma UcOCN levels were observed in spring than in summer. The plasma T₃ and T₄ levels were positively correlated with plasma UcOCN, whereas the plasma cortisol and prolactin were negatively correlated. Further, to observe the effect of season on temporal variations of UcOCN, Murrah buffaloes aged between 4–6 (n = 5), 14–16 (n = 5) and 28–30 (n = 5) months were bled at an interval of 30 min, between 0700 and 01030 h, during both the seasons. We observed significant effects of season (p < 0.0001), age (p < 0.0001) and season-by-age interaction (p < 0.01) on plasma UcOCN. The reasons for variations in plasma UcOCN with age and season will be discussed.     

Age-related changes and circadian variations in peripheral levels of thyroid hormones in Murrah buffaloes

The aim of this study was to investigate the variations in plasma triiodothyronine (T₃) and thyroxine (T₄) with the advancement of age and to determine their circadian patterns in prepubertal and pubertal Murrah buffaloes. The variations in plasma T₃ and T₄ with the advancement of age were observed from day 1 to 24 months of age. Significant higher levels of T₃ and T₄ were observed after birth and a gradual decrease in their concentrations occurred until 15 days of age. The mean plasma T₃ and T₄ ranged between 1.26–3.79 and 60.7–166 ng/ml, respectively, during 1–30 days of age. During 1–24 months of age, the variations in plasma T₃ did not differ (p > 0.05) with the advancement of age, whereas significant (p < 0.0001) changes were observed in plasma T₄. The circadian patterns of T₃ and T₄ were evaluated in prepubertal Murrah buffaloes (n = 8) aged between 14 and 16 months. The mean plasma T₃ and T₄ ranged between 1.04–1.85 and 43.0–76.1 ng/ml, respectively. Significant (p > 0.0001) changes in the secretory pattern of T₃ were observed, whereas the secretory pattern of T₄ did not differ significantly (p > 0.05). In addition, the circadian patterns of T₃ and T₄ in pubertal buffaloes (n = 4) aged between 28 and 30 months were observed and compared to that of prepubertal group (n = 4). The prepubertal group showed significant (p < 0.001) higher plasma T₃ concentrations over 24 h than the pubertal group.