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Oncogene amplification on extrachromosomal DNA (ecDNA) is strongly associated with treatment resistance and shorter survival for patients with cancer, including patients with glioblastoma. The non-chromosomal inheritance of ecDNA during cell division is a major contributor to intratumoral genetic heterogeneity. At present, the spatial dynamics of ecDNA, and the impact on tumor evolutionary trajectories, are not well understood. Here, we investigate the spatial-temporal evolution of ecDNA and its clinical impact by analyzing tumor samples from 94 treatment-naive human IDH -wildtype glioblastoma patients. We developed a spatial-temporal computational model of ecDNA positive tumors ('SPECIES') that integrates whole-genome sequencing, multi-region DNA FISH, and nascent RNAscope, to provide unique insight into the spatial dynamics of ecDNA evolution. Random segregation in combination with positive selection of ecDNAs induce large, predictable spatial patterns of cell-to-cell ecDNA copy number variation that are highly dependent on the oncogene encoded on the circular DNA. EGFR ecDNAs often reach high mean copy number (mean of 50 copies per tumor cell), are under strong positive selection (mean selection coefficient, s > 2) and do not co-amplify other oncogenes on the same ecDNA particles. In contrast, PDGFRA ecDNAs have lower mean copy number (mean of 15 copies per cell), are under weaker positive selection and frequently co-amplify other oncogenes on the same ecDNA. Evolutionary modeling suggests that EGFR ecDNAs often accumulate prior to clonal expansion. EGFR structural variants, including vIII and c-terminal deletions are under strong positive selection, are found exclusively on ecDNA, and are intermixed with wild-type EGFR ecDNAs. Simulations show EGFRvIII ecDNA likely arises after ecDNA formation in a cell with high wild-type EGFR copy number (> 10) before the onset of the most recent clonal expansion. This remains true even in cases of co-selection and co-amplification of multiple oncogenic ecDNA species in a subset of patients. Overall, our results suggest a potential time window in which early ecDNA detection may provide an opportunity for more effective intervention., Highlights: ecDNA is the most common mechanism of focal oncogene amplification in IDH wt glioblastoma. EGFR and its variants on ecDNA are particularly potent, likely arising early in tumor development, providing a strong oncogenic stimulus to drive tumorigenesis. Wild-type and variant EGFR ecDNA heteroplasmy (co-occurrence) is common with EGFR vIII or c-terminal deletions being derived from EGFR wild-type ecDNA prior to the most recent clonal expansion. Tumors with ecDNA amplified EGFR versus PDGFRA exhibit different evolutionary trajectories. SPECIES model can infer spatial evolutionary dynamics of ecDNA in cancer.A delay between ecDNA accumulation and subsequent oncogenic mutation may give a therapeutic window for early intervention.

Colorectal cancer (CRC) is a common malignancy worldwide and tumour stage is closely related to clinical outcome. A small but significant proportion of submucosal-invasive (ie, pT1) CRC are associated with regional lymph node metastases (LNM) and a worse prognosis. The likelihood of LNM in pT1 CRC needs to be balanced against the operative risk and costs of surgical resection when determining the best patient management. A wide range of histopathological and clinical factors may affect LNM risk in this setting. This script provides a comprehensive overview of the tumour and patient-associated features that have been linked to LNM risk in pT1 CRC. Some of the features are well established within the literature and are included in published guidelines, while others are novel and emerging in nature. Odds ratios for LNM that are associated with key predictive features are provided where appropriate, and published models developed as an aid to the calculation of LNM risk are discussed., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Benign Epilepsy with Centro-Temporal Spikes (BECTS) is a pediatric epilepsy with typically good seizure control. Although BECTS may increase patients' risk of developing neurological comorbidities, their clinical care and short-term outcomes are poorly quantified., Methods: We retrospectively assessed adherence to National Institute for Health and Care Excellence (NICE) guidelines relating to specialist referral, electroencephalogram (EEG) conduct and annual review in the care of patients with BECTS, and measured their seizure, neurodevelopmental and learning outcomes at three years post-diagnosis., Results: Across ten centers in England, we identified 124 patients (74 male) diagnosed with BECTS between 2015 and 2017. Patients had a mean age at diagnosis of 8.0 (95% CI = 7.6-8.4) years. 24/95 (25%) patients were seen by a specialist within two weeks of presentation; 59/100 (59%) received an EEG within two weeks of request; and 59/114 (52%) were reviewed annually. At three years post-diagnosis, 32/114 (28%) experienced ongoing seizures; 26/114 (23%) had reported poor school progress; 15/114 (13%) were diagnosed with a neurodevelopmental disorder (six autism spectrum disorder, six attention-deficit/hyperactivity disorder); and 10/114 (8.8%) were diagnosed with a learning difficulty (three processing deficit, three dyslexia). Center-level random effects models estimated neurodevelopmental diagnoses in 9% (95% CI: 2-16%) of patients and learning difficulty diagnoses in 7% (95% CI: 2-12%)., Conclusions: In this multicenter work, we found variable adherence to NICE guidelines in the care of patients with BECTS and identified a notable level of neurological comorbidity. Patients with BECTS may benefit from enhanced cognitive and behavioral assessment and monitoring., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shakti Agrawal has had speaking contracts with USB Medical, GW Pharmaceuticals and Nutricia. The remaining authors have no conflicts of interest to report., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Study Design: Systematic review., Background: Although degenerative cervical myelopathy (DCM) is the most prevalent spinal cord condition worldwide, the pathophysiology remains poorly understood. Our objective was to evaluate existing histological findings of DCM on cadaveric human spinal cord tissue and explore their consistency with animal models., Methods: MEDLINE and Embase were systematically searched (CRD42021281462) for primary research reporting on histological findings of DCM in human cadaveric spinal cord tissue. Data was extracted using a piloted proforma. Risk of bias was assessed using Joanna Briggs Institute critical appraisal tools. Findings were compared to a systematic review of animal models (Ahkter et al. 2020 Front Neurosci 14)., Results: The search yielded 4127 unique records. After abstract and full-text screening, 19 were included in the final analysis, reporting on 150 autopsies (71% male) with an average age at death of 67.3 years. All findings were based on haematoxylin and eosin (H&E) staining. The most commonly reported grey matter findings included neuronal loss and cavity formation. The most commonly reported white matter finding was demyelination. Axon loss, gliosis, necrosis and Schwann cell proliferation were also reported. Findings were consistent amongst cervical spondylotic myelopathy and ossification of the posterior longitudinal ligament. Cavitation was notably more prevalent in human autopsies compared to animal models., Conclusion: Few human spinal cord tissue studies have been performed. Neuronal loss, demyelination and cavitation were common findings. Investigating the biological basis of DCM is a critical research priority. Human spinal cord specimen may be an underutilised but complimentary approach., (© 2023. The Author(s).)

Traumatic brain injury is increasingly common in older individuals. Older age is one of the strongest predictors for poor prognosis after brain trauma, a phenomenon driven by the presence of extra-cranial comorbidities as well as pre-existent pathologies associated with cognitive impairment and brain volume loss (such as cerebrovascular disease or age-related neurodegeneration). Furthermore, ageing is associated with a dysregulated immune response, which includes attenuated responses to infection and vaccination, and a failure to resolve inflammation leading to chronic inflammatory states. In traumatic brain injury, where the immune response is imperative for the clearance of cellular debris and survey of the injured milieu, an appropriate self-limiting response is vital to promote recovery. Currently, our understanding of age-related factors that contribute to the outcome is limited; but a more complete understanding is essential for the development of tailored therapeutic strategies to mitigate the consequences of traumatic brain injury. Here we show greater functional deficits, white matter abnormalities and worse long-term outcomes in aged compared with young C57BL/6J mice after either moderate or severe traumatic brain injury. These effects are associated with altered systemic, meningeal and brain tissue immune response. Importantly, the impaired acute systemic immune response in the mice was similar to the findings observed in our clinical cohort. Traumatic brain-injured patient cohort over 70 years of age showed lower monocyte and lymphocyte counts compared with those under 45 years. In mice, traumatic brain injury was associated with alterations in peripheral immune subsets, which differed in aged compared with adult mice. There was a significant increase in transcription of immune and inflammatory genes in the meninges post-traumatic brain injury, including monocyte/leucocyte-recruiting chemokines. Immune cells were recruited to the region of the dural injury, with a significantly higher number of CD11b + myeloid cells in aged compared with the adult mice. In brain tissue, when compared with the young adult mice, we observed a more pronounced and widespread reactive astrogliosis 1 month after trauma in aged mice, sustained by an early and persistent induction of proinflammatory astrocytic state. These findings provide important insights regarding age-related exacerbation of neurological damage after brain trauma., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Objective: To explore how social media could be utilised to influence an individual's motivation to pursue a neurosurgical career, an emerging topic area. The focus of this study was on women interested in neurosurgery., Summary Background Data: Women are significantly under-represented in neurosurgery. 18% of all neurosurgeons - including 8% of consultants - are women. Most previous studies have used quantitative methods that are not best suited to gaining an in-depth understanding of the barriers that women face in pursuing a career in neurosurgery, or what would enable more women to go into the speciality., Methods: In this qualitative study, individual semi-structured interviews were conducted until data saturation was achieved. Participants were women pre-neurosurgical trainees. The interview data was examined through a thematic analysis involving open and axial coding., Results: Thirty women participated in the study. Four overarching themes were identified: (1) mentorship, (2) testimony from other women doing neurosurgery, (3) social media as a means of increasing interest in neurosurgery as a career choice, and (4) real-life exposure to the speciality., Conclusion: There is scope to further improve uptake of women into neurosurgical training in the UK. Motivations and barriers to women pursuing neurosurgery should be addressed openly through early experience, role models and mentorship. Social media can help facilitate these opportunities, disseminate information and inspiration, and has the potential to undo societal biases.

Introduction:  To explore our experience of hosting the 10 th  Annual Neurology and Neurosurgery Interest Group-Society of British Neurological Surgeons (NANSIG-SBNS) Neurosurgery Careers Day, held virtually for the first time., Methods:  Reflective feedback and review of an international, virtual neurosurgery careers day. The authors reflect on the logistics of organizing the event, and the pre- and post-event feedback provided by delegates. Recommendations have been made on how to successfully host a virtual event. The key themes that permeated the event have been outlined and discussed in the context of the feedback received., Results:  The event was attended by 231 delegates from 20 countries worldwide. Knowledge of neurosurgery as a career and the application process increased after attending the careers day (4.27/5 to 4.51/5, p=0.003 and 3.12/5 to 4.31/5, p<0.001 respectively). The key themes identified from the event include attendance, networking, and education. Qualitative feedback was positive and indicated a positive perception of the careers day., Conclusions:  The future of educational events is unclear, and a hybrid approach is recommended to retain the benefits of the online space when in-person events eventually return., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Richardson et al.)

Background: The adequacy of personal protective equipment (PPE) and infection prevention and control (IPC) training in UK medical students and interim Foundation Year 1 (FiY1) doctors during the COVID-19 pandemic is unknown, as is its impact on COVID-19-related anxiety., Methods: Cross-sectional, multi-centre study analysing self-reported adequacy of PPE and IPC training and correlation to a modified pandemic anxiety scale. Participants were current medical students and FiY1 doctors in the UK. Data were collected by an online survey., Results: Participants reported that they received insufficient PPE information (43%) and IPC training (56%). Significantly, fewer participants identifying as women or BAME/mixed ethnicity reported receiving sufficient PPE information, compared with those identifying as men and White British/White Other, respectively. COVID-19-related anxiety was significantly higher in those without sufficient reported PPE or IPC training, in women compared with men, and in FiY1 doctors compared with medical students., Conclusions: With medical students currently volunteering in and imminently returning to hospitals in an educational capacity, levels of self-reported PPE and IPC training are sub-optimal. Better training is paramount to avoid harm to patients and healthcare professionals and to reduce COVID-19-related anxiety among medical students and FiY1 doctors., (© Crown copyright 2020.)

Cerebral small vessel disease (cSVD) in penetrating arteries is a major cause of age-related morbidity. Cellular senescence is a molecular process targeted by novel senolytic drugs. We quantified senescence in penetrating arteries and tested whether myocyte senescence was associated with cSVD. We immunolabeled subcortical white matter of older persons (age 80-96 years, n = 60) with minimal AD, using antibodies to 2 established senescence markers (H3K9me3, γH2AX) and a myocyte marker (hSMM). Within the walls of penetrating arteries (20-300 µm), we quantified senescence-associated heterochromatic foci (SAHF)-positive nuclei, cell density (nuclei/µm2), and sclerotic index (SI). Senescent-appearing mural cells were present in small arteries of all cases. cSVD cases exhibited a lower proportion of senescent-appearing cells and lower area fraction (AF%) of SAHF-positive nuclei compared to controls (p = 0.014, 0.016, respectively). cSVD severity and SI both correlated negatively with AF% (p = 0.013, 0.002, respectively). Mural cell density was lower (p < 0.001) and SI higher (p < 0.001) in cSVD, relative to controls. In conclusion, senescent myocyte-like cells were universal in penetrating arteries of an AD-free cohort aged 80 years and older. Senescent-appearing nuclei were more common in persons aged 80 years and older without cSVD compared to cSVD cases, indicating caution in senolytic drug prescribing. Myocyte senescence and cSVD may represent alternative vessel fates in the aging human brain., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.

Background: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52., Methods and Results: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb., Conclusions: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Problem: The correlation of anti-sperm antibodies (ASA) with some instances of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as a more complete understanding of the mechanism behind this phenomenon, are dependent on the identification and characterization of relevant sperm antigens., Method of Study: In this article, we review literature on methods employed to identify sperm antigens using anti-sperm polyclonal and monoclonal antibodies from infertile patients and vasectomized men. Particular focus is given to approaches using human and mouse monoclonal antibodies to define the SAGA-1 human sperm antigen., Results: ASA present in sera and genital tract secretions from infertile patients and vasectomized men have been employed in a variety of methods to identify sperm antigens. In an alternate approach, a monoclonal antibody (mAb), H6-3C4, was immortalized from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titers. Subsequently, the sperm-agglutinating, murine S19 mAb was shown to react with the H6-3C4 cognate antigen. The H6-3C4 S19 cognate antigen, designated Sperm Agglutination Antigen-1 (SAGA-1), was characterized as a polymorphic, highly acidic, GPI-anchored glycoprotein on the surface of human spermatozoa. Purification with the S19 mAb followed by microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Immunoblot analysis demonstrated that these two glycoproteins differed in carbohydrate composition. Thus, sperm SAGA-1 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core peptide but with different carbohydrate structures., Conclusions: Autoimmunity to the SAGA-1 and/or CD52 glycoforms may lead to infertility. Structural and immunologic differences between these glycoproteins may be important factors in the etiology of immunologic infertility and other autoimmune disorders.

A major objective in developing a sperm antigen-based contraceptive vaccine for humans is the discovery of sperm surface immunogens that are functionally relevant and sperm specific. The latter criterion is deemed essential to avoid the possibility of inducing autoimmune disease upon vaccination. This review presents evidence that a unique carbohydrate epitope is synthesized in the human epididymis, is attached to the core peptide of CD52, a lymphocyte differentiation marker, and is subsequently inserted into the sperm membrane via a glycosylphosphatidylinositol anchor. This unique CD52 glycoform is localized to the entire sperm surface, functions as a potent target for agglutinating and cytotoxic antibodies, and is one of the few well-defined sperm surface glycoproteins indicated in human antibody-mediated infertility.

In a benchmark study, Isojima and colleagues established H6-3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6-3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15-25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6-3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.

The amino-acid sequence and three-dimensional structure of equine serum albumin have been determined. The amino-acid sequence was deduced from cDNA isolated from equine liver. Comparisons of the primary structure of equine serum albumin with human serum albumin and bovine serum albumin reveal 76.1% and 73.9% sequence identity, respectively. The three-dimensional structure was determined crystallographically by the molecular-replacement method using molecular coordinates from the previously determined structure of human serum albumin, to a resolution of 0.27 nm. In accordance with the primary structure, the three-dimensional structures are highly conserved. There is a root-mean-square difference between alpha-carbons of the two structures of 0.201 nm. The association constants (Ka) for the binding of 2,3,5-triiodobenzoic acid were determined by ultrafiltration methods for equine and human serum albumins to be approximately 10(4) M-1 and 10(5) M-1, respectively. Crystallographic studies of equine serum albumin reveal two binding sites for 2,3,5-triiodobenzoic acid identical with those previously reported for human serum albumin which are located within subdomains in IIA and IIIA. Details and comparisons of the binding chemistry are discussed.